This document discusses techniques for analyzing gene expression in formalin-fixed paraffin-embedded (FFPE) tissue samples using real-time PCR. FFPE samples are valuable for biomarker studies but RNA extracted from them is fragmented and damaged. The document presents a new method called RT2 FFPE PreAMP technology that combines improved RNA extraction and reverse transcription with a preamplification step to enhance detection of genes from FFPE samples in real-time PCR analysis. It allows for multi-gene expression profiling from archived clinical samples. Testing showed the method improved detection of genes and increased the positive call rate to 100% compared to standard methods.
This document describes an improved method for quantitative transcript profiling using cDNA-AFLP (cDNA amplified fragment length polymorphism). The key improvements allow it to be used as an efficient tool for genome-wide expression analysis as an alternative to microarrays. Unique transcript tags are generated from mRNA and screened through selective PCR amplifications. Based on in silico analysis, the enzyme combination BstYI and MseI was chosen to represent at least 60% of transcripts. The method was able to accurately detect differentially expressed genes and subtle expression differences. It was demonstrated to be useful by screening for cell cycle-modulated genes in tobacco.
The document summarizes the results of adding long read and linked read sequencing data to the Genome in a Bottle Consortium benchmark. Specifically:
- The benchmark regions now cover over 90% of the genome and include over 139,000 additional SNPs and 16,000 more insertions/deletions, mostly in difficult to sequence regions.
- The additional data improves variant detection in medically relevant genes and reduces errors compared to using short reads alone.
- However, performance of short read variant callers decreases when evaluated against the new expanded benchmark, highlighting remaining challenges in complex genomic regions.
This document describes a novel gene targeting approach called aptamer-guided gene targeting (AGT) that uses DNA aptamers selected through capillary electrophoresis systematic evolution of ligands by exponential enrichment to bind site-specific DNA binding proteins and guide donor DNA to specific genetic loci for correction. The approach was shown to increase gene targeting efficiency up to 32-fold in yeast and 16-fold in human cells. It also discusses the potential to develop aptamers for other genome editing tools like CRISPR/Cas9.
DNA Methylation: An Essential Element in Epigenetics Facts and TechnologiesQIAGEN
The document discusses DNA methylation and related epigenetic mechanisms. It defines epigenetics as the reversible inheritance of gene expression patterns not involving changes to DNA sequences. The key epigenetic mechanisms are described as DNA methylation, histone modification, and microRNA activity. DNA methylation involves the addition of a methyl group to cytosine bases in CpG dinucleotides and is an important regulator of gene expression. Methods for analyzing DNA methylation patterns like bisulfite sequencing are also summarized.
Accurate DNA Methylation Analysis with Successful Bisulfite Conversion WebinarQIAGEN
Bisulfite conversion is a popular used method for DNA methylation analysis. It is the most convenient and effective way to map DNA methylation to individual bases. The efficiency of the bisulfite conversion has a huge impact on the reliability of the downstream analysis methods and complete conversion is a prerequisite for correct determination of methylation. However, standard methods require harsh conditions with long incubations times at high temperatures and low pH to achieve complete conversion. This harsh treatment can lead to DNA degradation, lowering the yields and sensitivity of the subsequent analysis.
This slidedeck:
• Explains the principle of bisulfite conversion
• Points out the challenges and critical factors for successful bisulfite conversion
• Describes how to overcome the challenges with QIAGEN’s EpiTect Fast Bisulfite Kits
• Gives general recommendations for planning successful bisulfite conversion experiments
This study evaluated the feasibility of using 2' terminators and a DNA polymerase with reduced discrimination against unconventional nucleotides in the ViroSeq HIV genotyping assay as an alternative to the standard BigDye system. The results showed that the 2' terminator chemistry produced equivalent quality reads and genotyping results as the BigDye system. Removing the pyrophosphatase enzyme from the 2' terminator reactions appeared to improve signal quality. While read lengths were equivalent to BigDye, the alternative chemistry may provide benefits in cost and robustness and warrants further optimization and evaluation across different platforms and samples.
Polymerase chain reaction (PCR) is a common technique used to amplify a specific region of DNA, producing millions of copies. It uses the enzyme Taq polymerase to synthesize new DNA strands from existing DNA templates. The process involves repeated cycles of heating and cooling to denature and separate the DNA strands, allow primers to anneal, and extend new strands. This exponential process results in billions of copies of the target DNA region that can then be analyzed using gel electrophoresis or other techniques. PCR has many applications in research, forensics, genetic testing, and disease diagnosis.
This document describes an improved method for quantitative transcript profiling using cDNA-AFLP (cDNA amplified fragment length polymorphism). The key improvements allow it to be used as an efficient tool for genome-wide expression analysis as an alternative to microarrays. Unique transcript tags are generated from mRNA and screened through selective PCR amplifications. Based on in silico analysis, the enzyme combination BstYI and MseI was chosen to represent at least 60% of transcripts. The method was able to accurately detect differentially expressed genes and subtle expression differences. It was demonstrated to be useful by screening for cell cycle-modulated genes in tobacco.
The document summarizes the results of adding long read and linked read sequencing data to the Genome in a Bottle Consortium benchmark. Specifically:
- The benchmark regions now cover over 90% of the genome and include over 139,000 additional SNPs and 16,000 more insertions/deletions, mostly in difficult to sequence regions.
- The additional data improves variant detection in medically relevant genes and reduces errors compared to using short reads alone.
- However, performance of short read variant callers decreases when evaluated against the new expanded benchmark, highlighting remaining challenges in complex genomic regions.
This document describes a novel gene targeting approach called aptamer-guided gene targeting (AGT) that uses DNA aptamers selected through capillary electrophoresis systematic evolution of ligands by exponential enrichment to bind site-specific DNA binding proteins and guide donor DNA to specific genetic loci for correction. The approach was shown to increase gene targeting efficiency up to 32-fold in yeast and 16-fold in human cells. It also discusses the potential to develop aptamers for other genome editing tools like CRISPR/Cas9.
DNA Methylation: An Essential Element in Epigenetics Facts and TechnologiesQIAGEN
The document discusses DNA methylation and related epigenetic mechanisms. It defines epigenetics as the reversible inheritance of gene expression patterns not involving changes to DNA sequences. The key epigenetic mechanisms are described as DNA methylation, histone modification, and microRNA activity. DNA methylation involves the addition of a methyl group to cytosine bases in CpG dinucleotides and is an important regulator of gene expression. Methods for analyzing DNA methylation patterns like bisulfite sequencing are also summarized.
Accurate DNA Methylation Analysis with Successful Bisulfite Conversion WebinarQIAGEN
Bisulfite conversion is a popular used method for DNA methylation analysis. It is the most convenient and effective way to map DNA methylation to individual bases. The efficiency of the bisulfite conversion has a huge impact on the reliability of the downstream analysis methods and complete conversion is a prerequisite for correct determination of methylation. However, standard methods require harsh conditions with long incubations times at high temperatures and low pH to achieve complete conversion. This harsh treatment can lead to DNA degradation, lowering the yields and sensitivity of the subsequent analysis.
This slidedeck:
• Explains the principle of bisulfite conversion
• Points out the challenges and critical factors for successful bisulfite conversion
• Describes how to overcome the challenges with QIAGEN’s EpiTect Fast Bisulfite Kits
• Gives general recommendations for planning successful bisulfite conversion experiments
This study evaluated the feasibility of using 2' terminators and a DNA polymerase with reduced discrimination against unconventional nucleotides in the ViroSeq HIV genotyping assay as an alternative to the standard BigDye system. The results showed that the 2' terminator chemistry produced equivalent quality reads and genotyping results as the BigDye system. Removing the pyrophosphatase enzyme from the 2' terminator reactions appeared to improve signal quality. While read lengths were equivalent to BigDye, the alternative chemistry may provide benefits in cost and robustness and warrants further optimization and evaluation across different platforms and samples.
Polymerase chain reaction (PCR) is a common technique used to amplify a specific region of DNA, producing millions of copies. It uses the enzyme Taq polymerase to synthesize new DNA strands from existing DNA templates. The process involves repeated cycles of heating and cooling to denature and separate the DNA strands, allow primers to anneal, and extend new strands. This exponential process results in billions of copies of the target DNA region that can then be analyzed using gel electrophoresis or other techniques. PCR has many applications in research, forensics, genetic testing, and disease diagnosis.
The document describes a seminar on Random Amplified Polymorphic DNA (RAPD) markers. It defines RAPD as a type of PCR reaction that amplifies random segments of DNA using short arbitrary nucleotide primers. The document outlines the history, principle, procedure, applications, advantages, and limitations of RAPD analysis. It compares RAPD to other molecular marker techniques and concludes that RAPD is a lab technique used to amplify unknown DNA segments for analysis.
This document discusses various methods for normalization of RNA-seq read count data, including RPKM/FPKM, TMM, Limma voom, and TPM. It provides explanations of each method and how they aim to correct for differences in sequencing depth, transcript length, and transcript pool composition between samples. The document also provides examples of calculating RPKM, TPM, and comparing the two methods. Lastly, it discusses using tools like RSEM, Bowtie, and EBSeq for determining differentially expressed genes from RNA-seq data through a count-based strategy.
1. Pyrosequencing is a DNA sequencing technique based on detecting pyrophosphate release upon nucleotide incorporation, unlike Sanger sequencing which uses chain termination.
2. In pyrosequencing, only one of the four possible nucleotides is added at a time so light emission determines which nucleotide is incorporated on the template.
3. Next generation sequencing includes 454 pyrosequencing which was the first commercially successful technique and works by parallelizing pyrosequencing on many DNA fragments attached to beads in a picotiter plate.
DNA amplification techniques include in vivo cloning and in vitro PCR. PCR was independently proposed in the 1970s and 1980s and allows selective amplification of DNA segments using a thermostable DNA polymerase. Key components of PCR include a template DNA, primers, DNA polymerase, nucleotides, and magnesium. During cycling, the DNA is denatured, primers anneal, and the polymerase extends the DNA. PCR has revolutionized molecular biology due to its ability to rapidly amplify specific DNA regions.
An Investigation Of The Rigor Of Interpretation RulesNick Brown
1. The authors analyzed DNA samples ranging from 1 ng to 0.8 pg using STR profiling with varying numbers of PCR cycles. They found that 34 cycles provided full profiles down to 25-50 pg with minimal allele dropout or imbalance, and no advantage to using more cycles.
2. Even under stringent laboratory conditions, some level of contamination was observed, with 21 of 30 negative controls showing low-level spurious alleles. Replicate analyses of a 3 pg sample showed 6 contaminant alleles.
3. Guidelines for interpreting low-template DNA profiles were developed based on duplication of results. A statistical model was also developed to model dropout, contamination, and stutter to evaluate the reliability of the guidelines.
1) Discovery of over 1 million structural variant calls from 30+ sequence-resolved callsets across 4 technologies for an AJ Trio. 2) After clustering, over 128,000 sequence-resolved SV calls >=50bp remained. 3) Over 30,000 SVs had support from 2+ technologies or 5+ callers with sequences <20% different or support from optical mapping.
Site-directed mutagenesis is a molecular biology technique used to make specific changes to DNA sequences. It involves using a primer containing the desired mutation in a PCR reaction to introduce the mutation into the gene of interest. There are different approaches for site-directed mutagenesis using PCR, including using a mutated primer in normal PCR or a primer extension method. The technique is used for applications like protein engineering to study the impact of sequence changes or insert restriction sites. However, it can be difficult to replicate the mutated DNA and screening mutations requires sequencing.
Target enrichment enables researchers to focus their next generation sequencing (NGS) efforts on regions of interest, allowing them to obtain more sequencing data relevant to their study. In-solution target capture is a method of enrichment using oligonucleotide probes directed to specific regions within a genome. Target capture can be used to enrich multiple samples simultaneously, reducing the cost per sample, while using individually synthesized probes allows researchers to construct gene panels that can be optimized over time.
This document discusses several types of PCR techniques and their applications. It begins by explaining standard PCR and its development. It then describes several specialized PCR techniques including allele-specific PCR, asymmetric PCR, assembly PCR, hot-start PCR, helicase-dependent amplification, in situ PCR, inverse PCR, ligation-mediated PCR, and multiplex ligation-dependent probe amplification. Each technique is explained and examples of its uses and applications are provided.
This document summarizes two methods for mapping DNA-protein interactions: DNase I footprinting and DMS footprinting. DNase I footprinting involves digesting DNA with DNase I after protein binding, which will be protected by the protein. DMS footprinting uses dimethyl sulfate to modify purines, which will be protected by bound protein. The document also reviews mechanisms of regulation of the lac and tryptophan operons, including activation of lac by cAMP-CAP and attenuation control of tryptophan based on tryptophan levels.
Molecular marker technology in studies on plant genetic diversityChanakya P
A molecular marker is a molecule contained within a sample taken from an organism (biological markers) or other matter. It can be used to reveal certain characteristics about the respective source. DNA, for example, is a molecular marker containing information about genetic disorders, genealogy and the evolutionary history of life. Specific regions of the DNA (genetic markers) are used to diagnose the autosomal recessive genetic disorder cystic fibrosis, taxonomic affinity (phylogenetics) and identity (DNA Barcoding). Further, life forms are known to shed unique chemicals, including DNA, into the environment as evidence of their presence in a particular location.Other biological markers, like proteins, are used in diagnostic tests for complex neurodegenerative disorders, such as Alzheimer's disease. Non-biological molecular markers are also used, for example, in environmental studies.
1. There are several types of polymerase chain reaction (PCR) that have been developed to amplify DNA sequences for various purposes. These include inverse PCR, multiplex PCR, nested PCR, and ligation-mediated PCR.
2. Inverse PCR uses restriction enzymes and circularization to amplify unknown DNA sequences flanking a known region. Multiplex PCR allows for amplification of multiple targets simultaneously. Nested PCR uses two rounds of PCR with nested primers for increased specificity. Ligation-mediated PCR ligates linkers to DNA fragments before amplifying them.
3. These specialized PCR techniques have various applications in forensics, genetics, molecular biology research, and medicine. They allow researchers to study DNA sequences in new
PCR is a technique used to amplify a specific DNA sequence. It involves three basic steps - denaturation of DNA, annealing of primers to the DNA templates, and extension of the primers by DNA polymerase. The amplified DNA can then be analyzed for various applications in medicine, forensics, and research. Some key advantages of PCR are that it is automated, fast, reliable, sensitive and has a high output.
This presentation summarizes three topics: polymerase chain reaction (PCR), antisense therapy, and the complement system. PCR is an in vitro technique used to amplify a specific DNA sequence and was invented by Kary Mullis in 1983. It involves DNA denaturation, annealing of primers, and extension of the DNA sequence. Applications include genome mapping, disease diagnosis, and forensics. Antisense therapy inhibits gene expression by using single-stranded oligonucleotides to block transcription. It is used to treat various cancers and infections. The complement system consists of serum proteins that help activate the innate immune response. It can be activated via the classical or alternative pathway and acts through mechanisms like opsonization, inflammation, and
Watch the presentation of this webinar here: https://bit.ly/2SWCycq
mRNA has taken center stage. Vaccines and therapeutics based on this versatile biomolecule have the potential to transform disease prevention and treatment. This webinar will explore key considerations for efficient mRNA production, starting from facility design and raw materials selection to technologies and strategies used for manufacturing.
The success of mRNA-based COVID-19 vaccines has created a significant level of interest in this versatile biomolecule for disease prevention and treatment. While production of these vaccines took place in record time, critical decisions must be made when developing novel mRNA applications to ensure manufacturability, reproducibility, and safety. This webinar will explore foundational elements of the mRNA manufacturing workflow and strategies to design the right facilities to ensure success. Topics include collaborative approaches to ensure access to high quality raw materials, application of advanced technologies for manufacturing, options for facility design and key considerations when leveraging a contract development and manufacturing partner.
In this webinar, you will learn:
• Therapeutic potential of mRNA: COVID-19 and beyond
• How mRNA manufacturing workflows and facility design have a significant impact on reproducibility and performance
• Amptec capabilities to accelerate mRNA development and manufacturing
The document discusses the polymerase chain reaction (PCR) technique. It describes how PCR can amplify a small number of DNA copies into thousands or millions of copies. The key components of PCR include the target DNA, primers, dNTPs, thermostable DNA polymerase, Mg2+ ions, and a buffer solution. The document also outlines several applications of PCR in agriculture, including product development, grain processing, fishery product identification, cultivar identification of rice, and quantification of Fusarium culmorum in wheat and barley. Finally, it states that PCR is often used to detect products of agricultural biotechnology and has revolutionized molecular biology.
Addressing the Pre-PCR Analytical Variability of FFPE SamplesCandy Smellie
Despite technical advances, assessing the accuracy of pre-PCR steps, which include DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues, DNA quantitation and DNA quality control, remain a key challenge in external quality assurance.
In the webinar we will discuss the latest results from recent studies and look at ways that the accuracy of pre-PCR workflows can be improved.
Molecular methods of diagnosing infectious diseaseaka_sam15
Molecular methods such as PCR and LAMP have revolutionized infectious disease diagnosis by allowing rapid and sensitive detection of pathogens. PCR amplifies specific DNA sequences, and real-time PCR with fluorescent probes like TaqMan or molecular beacons allows quantification during amplification. LAMP is an inexpensive isothermal method that amplifies DNA with high sensitivity and specificity using multiple primers and strand displacement. Both PCR and LAMP have advanced diagnosis by detecting pathogens earlier and multiplexing the detection of multiple targets in a single sample.
1. Molecular genetics is the study of genetic makeup at the DNA level and overlaps with genetics, biochemistry, and other biological fields.
2. Advances in molecular genetics techniques like molecular markers, PCR, and genotyping allow for early and non-invasive selection as well as analysis of genetic diversity in poultry.
3. Common molecular marker techniques discussed are RFLP, RAPD, microsatellites, AFLP, and SNPs, which can be used for applications like parentage determination, gene mapping, and marker-assisted selection.
The pragmatic text miner: It's just another type of poorly standardized dataLars Juhl Jensen
The document discusses text mining and its uses, including summarizing unstructured biomedical literature and integrating various data sources. It addresses challenges like different data formats, identifiers, and quality. Collaboration with domain experts is important to determine what topics to analyze and how best to solve problems. Text mining can help curate knowledge from experimental data, predictions, and online resources about proteins, chemicals, subcellular localization, tissue expression, and disease associations.
The document discusses the benefits of exercise for mental health. Regular physical activity can help reduce anxiety and depression and improve mood and cognitive functioning. Exercise causes chemical changes in the brain that may help protect against mental illness and improve symptoms.
The document describes a seminar on Random Amplified Polymorphic DNA (RAPD) markers. It defines RAPD as a type of PCR reaction that amplifies random segments of DNA using short arbitrary nucleotide primers. The document outlines the history, principle, procedure, applications, advantages, and limitations of RAPD analysis. It compares RAPD to other molecular marker techniques and concludes that RAPD is a lab technique used to amplify unknown DNA segments for analysis.
This document discusses various methods for normalization of RNA-seq read count data, including RPKM/FPKM, TMM, Limma voom, and TPM. It provides explanations of each method and how they aim to correct for differences in sequencing depth, transcript length, and transcript pool composition between samples. The document also provides examples of calculating RPKM, TPM, and comparing the two methods. Lastly, it discusses using tools like RSEM, Bowtie, and EBSeq for determining differentially expressed genes from RNA-seq data through a count-based strategy.
1. Pyrosequencing is a DNA sequencing technique based on detecting pyrophosphate release upon nucleotide incorporation, unlike Sanger sequencing which uses chain termination.
2. In pyrosequencing, only one of the four possible nucleotides is added at a time so light emission determines which nucleotide is incorporated on the template.
3. Next generation sequencing includes 454 pyrosequencing which was the first commercially successful technique and works by parallelizing pyrosequencing on many DNA fragments attached to beads in a picotiter plate.
DNA amplification techniques include in vivo cloning and in vitro PCR. PCR was independently proposed in the 1970s and 1980s and allows selective amplification of DNA segments using a thermostable DNA polymerase. Key components of PCR include a template DNA, primers, DNA polymerase, nucleotides, and magnesium. During cycling, the DNA is denatured, primers anneal, and the polymerase extends the DNA. PCR has revolutionized molecular biology due to its ability to rapidly amplify specific DNA regions.
An Investigation Of The Rigor Of Interpretation RulesNick Brown
1. The authors analyzed DNA samples ranging from 1 ng to 0.8 pg using STR profiling with varying numbers of PCR cycles. They found that 34 cycles provided full profiles down to 25-50 pg with minimal allele dropout or imbalance, and no advantage to using more cycles.
2. Even under stringent laboratory conditions, some level of contamination was observed, with 21 of 30 negative controls showing low-level spurious alleles. Replicate analyses of a 3 pg sample showed 6 contaminant alleles.
3. Guidelines for interpreting low-template DNA profiles were developed based on duplication of results. A statistical model was also developed to model dropout, contamination, and stutter to evaluate the reliability of the guidelines.
1) Discovery of over 1 million structural variant calls from 30+ sequence-resolved callsets across 4 technologies for an AJ Trio. 2) After clustering, over 128,000 sequence-resolved SV calls >=50bp remained. 3) Over 30,000 SVs had support from 2+ technologies or 5+ callers with sequences <20% different or support from optical mapping.
Site-directed mutagenesis is a molecular biology technique used to make specific changes to DNA sequences. It involves using a primer containing the desired mutation in a PCR reaction to introduce the mutation into the gene of interest. There are different approaches for site-directed mutagenesis using PCR, including using a mutated primer in normal PCR or a primer extension method. The technique is used for applications like protein engineering to study the impact of sequence changes or insert restriction sites. However, it can be difficult to replicate the mutated DNA and screening mutations requires sequencing.
Target enrichment enables researchers to focus their next generation sequencing (NGS) efforts on regions of interest, allowing them to obtain more sequencing data relevant to their study. In-solution target capture is a method of enrichment using oligonucleotide probes directed to specific regions within a genome. Target capture can be used to enrich multiple samples simultaneously, reducing the cost per sample, while using individually synthesized probes allows researchers to construct gene panels that can be optimized over time.
This document discusses several types of PCR techniques and their applications. It begins by explaining standard PCR and its development. It then describes several specialized PCR techniques including allele-specific PCR, asymmetric PCR, assembly PCR, hot-start PCR, helicase-dependent amplification, in situ PCR, inverse PCR, ligation-mediated PCR, and multiplex ligation-dependent probe amplification. Each technique is explained and examples of its uses and applications are provided.
This document summarizes two methods for mapping DNA-protein interactions: DNase I footprinting and DMS footprinting. DNase I footprinting involves digesting DNA with DNase I after protein binding, which will be protected by the protein. DMS footprinting uses dimethyl sulfate to modify purines, which will be protected by bound protein. The document also reviews mechanisms of regulation of the lac and tryptophan operons, including activation of lac by cAMP-CAP and attenuation control of tryptophan based on tryptophan levels.
Molecular marker technology in studies on plant genetic diversityChanakya P
A molecular marker is a molecule contained within a sample taken from an organism (biological markers) or other matter. It can be used to reveal certain characteristics about the respective source. DNA, for example, is a molecular marker containing information about genetic disorders, genealogy and the evolutionary history of life. Specific regions of the DNA (genetic markers) are used to diagnose the autosomal recessive genetic disorder cystic fibrosis, taxonomic affinity (phylogenetics) and identity (DNA Barcoding). Further, life forms are known to shed unique chemicals, including DNA, into the environment as evidence of their presence in a particular location.Other biological markers, like proteins, are used in diagnostic tests for complex neurodegenerative disorders, such as Alzheimer's disease. Non-biological molecular markers are also used, for example, in environmental studies.
1. There are several types of polymerase chain reaction (PCR) that have been developed to amplify DNA sequences for various purposes. These include inverse PCR, multiplex PCR, nested PCR, and ligation-mediated PCR.
2. Inverse PCR uses restriction enzymes and circularization to amplify unknown DNA sequences flanking a known region. Multiplex PCR allows for amplification of multiple targets simultaneously. Nested PCR uses two rounds of PCR with nested primers for increased specificity. Ligation-mediated PCR ligates linkers to DNA fragments before amplifying them.
3. These specialized PCR techniques have various applications in forensics, genetics, molecular biology research, and medicine. They allow researchers to study DNA sequences in new
PCR is a technique used to amplify a specific DNA sequence. It involves three basic steps - denaturation of DNA, annealing of primers to the DNA templates, and extension of the primers by DNA polymerase. The amplified DNA can then be analyzed for various applications in medicine, forensics, and research. Some key advantages of PCR are that it is automated, fast, reliable, sensitive and has a high output.
This presentation summarizes three topics: polymerase chain reaction (PCR), antisense therapy, and the complement system. PCR is an in vitro technique used to amplify a specific DNA sequence and was invented by Kary Mullis in 1983. It involves DNA denaturation, annealing of primers, and extension of the DNA sequence. Applications include genome mapping, disease diagnosis, and forensics. Antisense therapy inhibits gene expression by using single-stranded oligonucleotides to block transcription. It is used to treat various cancers and infections. The complement system consists of serum proteins that help activate the innate immune response. It can be activated via the classical or alternative pathway and acts through mechanisms like opsonization, inflammation, and
Watch the presentation of this webinar here: https://bit.ly/2SWCycq
mRNA has taken center stage. Vaccines and therapeutics based on this versatile biomolecule have the potential to transform disease prevention and treatment. This webinar will explore key considerations for efficient mRNA production, starting from facility design and raw materials selection to technologies and strategies used for manufacturing.
The success of mRNA-based COVID-19 vaccines has created a significant level of interest in this versatile biomolecule for disease prevention and treatment. While production of these vaccines took place in record time, critical decisions must be made when developing novel mRNA applications to ensure manufacturability, reproducibility, and safety. This webinar will explore foundational elements of the mRNA manufacturing workflow and strategies to design the right facilities to ensure success. Topics include collaborative approaches to ensure access to high quality raw materials, application of advanced technologies for manufacturing, options for facility design and key considerations when leveraging a contract development and manufacturing partner.
In this webinar, you will learn:
• Therapeutic potential of mRNA: COVID-19 and beyond
• How mRNA manufacturing workflows and facility design have a significant impact on reproducibility and performance
• Amptec capabilities to accelerate mRNA development and manufacturing
The document discusses the polymerase chain reaction (PCR) technique. It describes how PCR can amplify a small number of DNA copies into thousands or millions of copies. The key components of PCR include the target DNA, primers, dNTPs, thermostable DNA polymerase, Mg2+ ions, and a buffer solution. The document also outlines several applications of PCR in agriculture, including product development, grain processing, fishery product identification, cultivar identification of rice, and quantification of Fusarium culmorum in wheat and barley. Finally, it states that PCR is often used to detect products of agricultural biotechnology and has revolutionized molecular biology.
Addressing the Pre-PCR Analytical Variability of FFPE SamplesCandy Smellie
Despite technical advances, assessing the accuracy of pre-PCR steps, which include DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues, DNA quantitation and DNA quality control, remain a key challenge in external quality assurance.
In the webinar we will discuss the latest results from recent studies and look at ways that the accuracy of pre-PCR workflows can be improved.
Molecular methods of diagnosing infectious diseaseaka_sam15
Molecular methods such as PCR and LAMP have revolutionized infectious disease diagnosis by allowing rapid and sensitive detection of pathogens. PCR amplifies specific DNA sequences, and real-time PCR with fluorescent probes like TaqMan or molecular beacons allows quantification during amplification. LAMP is an inexpensive isothermal method that amplifies DNA with high sensitivity and specificity using multiple primers and strand displacement. Both PCR and LAMP have advanced diagnosis by detecting pathogens earlier and multiplexing the detection of multiple targets in a single sample.
1. Molecular genetics is the study of genetic makeup at the DNA level and overlaps with genetics, biochemistry, and other biological fields.
2. Advances in molecular genetics techniques like molecular markers, PCR, and genotyping allow for early and non-invasive selection as well as analysis of genetic diversity in poultry.
3. Common molecular marker techniques discussed are RFLP, RAPD, microsatellites, AFLP, and SNPs, which can be used for applications like parentage determination, gene mapping, and marker-assisted selection.
The pragmatic text miner: It's just another type of poorly standardized dataLars Juhl Jensen
The document discusses text mining and its uses, including summarizing unstructured biomedical literature and integrating various data sources. It addresses challenges like different data formats, identifiers, and quality. Collaboration with domain experts is important to determine what topics to analyze and how best to solve problems. Text mining can help curate knowledge from experimental data, predictions, and online resources about proteins, chemicals, subcellular localization, tissue expression, and disease associations.
The document discusses the benefits of exercise for mental health. Regular physical activity can help reduce anxiety and depression and improve mood and cognitive functioning. Exercise causes chemical changes in the brain that may help protect against mental illness and improve symptoms.
Protein networks: A basis for large-scale data miningLars Juhl Jensen
The document discusses protein networks and how they can be used as a basis for large-scale data mining. It describes three phases: 1) constructing association networks using various data types, 2) building signaling networks by identifying signaling events and sequence motifs, and 3) examining dynamic networks to understand when interactions occur, such as during cell cycle regulation. The goal is to integrate different data sources to generate specific predictions and gain insights into systems properties and functions.
La producción de semillas en la provincia de Bolívar: un enfoque hacia el cam...HELP YOU ECUADOR
Este documento describe los esfuerzos para promover el acceso y uso de semillas de calidad entre los productores de la provincia de Bolívar en Ecuador. Se adquirieron semillas certificadas de papa, maíz, frijol, trigo y quinua para distribuir a organizaciones de productores. Los resultados mostraron que las organizaciones produjeron semillas con rendimientos altos y extrajeron un promedio de 52,7% de semilla para la próxima siembra. Sin embargo, todavía existe una demanda insatisfecha de semillas de calidad en la prov
DNUG 2008 – Unlocking the Business value of Notes & Domino data – Goodyear Du...Synaptris Inc.
This presentation made by Goodyear at the DNUG June 2008 Conference, explains the two most common ways used by them to provide users of Notes applications with information - (a) Writing scripts to create reports or extract data from Notes databases and (b) Exporting Notes data to external tools such as spreadsheets, and performing extensive manual operations to format data & generate reports.
Stefan Kirsch and Ralf Niedling from Goodyear discuss the challenges they faced with the above methods and elaborate the approaches and tools that they evaluated to address their need to maximize the business value of their Notes & Domino data. The presentation outlines how Goodyear was able to effectively meet their business and IT objectives using IntelliPRINT Reporting and the specific business efficiency gains that they have been able to achieve with the deployment of IntelliPRINT Reporting.
Read more about IntelliPRINT Reporting at - http://www.synaptris.com/intelliprint
Read this full article at - http://www.synaptris.com/news/2008/intelliprint_goodyear_dunlop_at_dnug.html
This document outlines the course content for a cell biology course. It covers 10 main topics: introduction to cells, chemical foundations, methods of studying cells, genetic mechanisms, cell signaling, cell membranes and architecture, energetics, cellular traffic, cell birth/lineage/death, and the molecular basis of cancer. The course will involve seminar presentations by students on each topic, along with exams to assess comprehension. Overall, the course provides an introduction to the key concepts and components of cell biology from a biochemical and genetic perspective.
Rice is a grain that comes from plants in the grass family. It is native to tropical and subtropical Asia and Africa. Rice plants grow 2-6 feet tall and produce grains that provide over one-fifth of human calories globally. Rice comes in thousands of varieties and is one of the most important human foods, sustaining two-thirds of the world's population. Small-scale farmers produce about four-fifths of the world's rice for local consumption.
This document discusses open access literature and its importance. Open access literature refers to digital content that is available online for free without restrictions. It is important because subscription costs for scholarly journals continue rising each year, limiting access. While research is funded by taxpayers and students, they must then pay high costs to access published results. Open access addresses this by removing barriers to access. It benefits students, researchers and libraries by providing free scholarly content. The document outlines how librarians and faculty can promote open access through publishing in and advocating for open access journals and repositories.
2013 ClinicalKey Quick User Guide & Specialty Content Listelsevierkoreahs
한편, ClinicalKey는 2012년 4월에 론칭된 임상 의학 정보 데이터베이스로서, 의학 및 수술과 관련된 방대한 임상 정보로부터 의사가 원하는 정확한 정보를 가장 빠르게 검색해 주는 차세대 임상 검색 엔진이다.
특히 검색 엔진 내에 900종 이상의 의학서적(eBook)과 500종 이상의 의학 저널, 이미지 및 13,000여개 이상의 수술 동영상을 포함하고 있어, 임상뿐만 아니라 학문적으로도 가장 관련성이 높은 근거중심(Evidence-based) 답변을 제공한다. 또한 전문가 논평, MEDLINE 초록 및 다른 출판사의 선택된 저널도 하나의 플랫폼에서 검색이 가능하여, 의사들의 정보검색 시간을 대폭 감소시킴으로써, 보다 더 많은 시간을 환자치료에 사용할 수 있도록 설계되었다.
This document outlines the course content for a cell biology course. It covers 10 main topics: introduction to cells, chemical foundations, methods of studying cells, genetic mechanisms, cell signaling, cell membranes and architecture, energetics, cellular traffic, cell birth and death, and the molecular basis of cancer. The course will involve seminar presentations by students on each topic, along with exams to assess comprehension. Overall, the document provides an overview of the key concepts and areas to be covered in an introductory cell biology course.
This study characterized two sporulation-specific kinases, CPE0213 and CPE1754, in Clostridium perfringens. RT-PCR showed that both kinases are transcriptionally active under sporulation conditions. Mutants lacking either kinase gene showed significantly reduced sporulation frequencies compared to wild type. Complementation experiments were unsuccessful due to overexpression issues. Future work will characterize the other four candidate kinases and perform in vitro phosphorylation assays.
High-throughput processing to maximize genomic analysis through simultaneous ...Thermo Fisher Scientific
As personalized cancer care evolves, the patient’s nucleic acid becomes ever so important to provide valuable information regarding their genetic makeup and disease state. Common sample types for these analyses include biopsies, which can be very limited in material making the downstream measurement of more than one analyte rather difficult. Obtaining another biopsy, using a different section or splitting the sample can be problematic because of tumor heterogeneity. Even adjacent areas of the same tumor tissue can result in different RNA/DNA profiles so the ability to isolate multiple analytes from the same sample offer a number of benefits, which include preserving samples and data consistency eliminating any sample to sample variation. As more tests are developed to simultaneously monitor genetic alterations, there is a strong need to efficiently isolate both DNA and RNA from the same starting sample in a format compatible with high-throughput processing.
Simultaneous Isolation of RNA & DNA from one FFPE SampleQIAGEN
The document summarizes the AllPrep DNA/RNA FFPE Kit, which simultaneously isolates genomic DNA and total RNA from formalin-fixed paraffin-embedded (FFPE) tissue samples. FFPE samples are lysed and centrifuged to separate the RNA-containing supernatant from the DNA-containing pellet. The supernatant and pellet are then processed separately to purify high-quality RNA and DNA suitable for downstream applications like real-time PCR and pyrosequencing. Results show the kit yields RNA and DNA of sufficient quality and quantity from old FFPE samples for gene expression analysis and detection of genetic mutations.
This document provides the timetable and protocols for a practical course on making and analyzing tRNA synthetases in vivo and using cell-free protein synthesis. Over two weeks, students will perform site-directed mutagenesis to produce mutant aminoacyl-tRNA synthetases, express their proteins in E. coli cells and purify the proteins, and use cell-free synthesis to attempt incorporating a phosphotyrosine analogue into a target protein using their mutant synthetases and suppressor tRNA. The document outlines the experimental steps, including mutagenesis, transformation, plasmid preparation, sequencing, protein expression and purification, cell-free reaction set up, and analysis by SDS-PAGE. Safety procedures are also described to handle
The Genome in a Bottle (GIAB) project provides reference materials and benchmarks for validating genome sequencing and variant calling. It has characterized variants in five human genomes, including common and difficult variants. While it currently enables benchmarking of easier variants, GIAB is working to characterize more difficult variants and regions. Many challenges remain in benchmarking structural variants and regions with lower confidence, and collaborations are welcome to help address these challenges.
The document provides an overview of polymerase chain reaction (PCR) techniques. It begins with an introduction to molecular biology techniques and the importance of hands-on experience. It then describes several key molecular techniques including PCR, gel electrophoresis, northern blotting, and southern blotting. The bulk of the document focuses on describing PCR in detail, including its history, components, steps, types, applications, advantages, and limitations. It also briefly discusses gel electrophoresis and provides an overview of the northern blotting process.
Comparing Mutation Detection Sensitivity from Matched FFPE Tissue and Liquid ...Thermo Fisher Scientific
Cancer researchers are avidly working to enable circulating cell free DNA (cfDNA) profiling as a new more sensitive tool to detect and screen for the presence of solid tumors before detection through clinical methods. Despite the high level of interest in cfDNA, researchers still have reservations until enough data has demonstrated complementarity between methodologies. In this study, we examined the data quality and concordance of mutations called for a small number of matched formalin fixed paraffin embedded (FFPE) tissue and plasma samples.
This document summarizes an experiment using the pAcGFP1-Mem vector to transform E. coli cells and visualize GFP localization to cellular membranes. Key findings include:
1) Gel electrophoresis and spectrometer data confirmed successful transformation of the pAcGFP1-Mem vector into E. coli genomes.
2) Confocal microscopy images clearly showed GFP localization to cellular membranes, with some fluorescence also seen in intracellular membranes.
3) While the pAcGFP1-Mem vector reliably highlights extracellular membranes, its ability to also label some intracellular membranes could be seen as an advantage for experiments examining both membrane types.
4) Overall, the results indicate the pAcGFP1-Mem vector is a simple
Cancer is a disease characterized by uncontrolled cell growth and proliferation. Recent advances in molecular medicine and cancer biology have changed the way research clinicians evaluate and consider treatment. Selected tumor biomarkers have been utilized as targets for drug therapy leading to better more effective treatment. Gene expression profiling has been used for identifying new biomarkers for tumor classification and driving decision making for better patient outcome in different tumor types. DNA microarrays have become a key method to acquire a comparative snapshot of the gene expression profile from test samples in a high throughput manner. Quantitative PCR and newer sequencing techniques are popular research alternatives offering highly accurate gene expression measurements, but with limitations due to cost, complex instrumentation and analysis needs. RNA extracted from formalin fixed paraffin embedded tissue (FFPE) creates considerable additional challenges in acquiring accurate gene expression measurements due to the highly fragmented and compromised integrity of FFPE RNA due to the fixation process.
The document summarizes the steps taken in a cloning simulation project to clone the erythropoietin gene. Key steps included obtaining the gene sequence from GenBank, analyzing it to find the ORF and restriction sites, designing primers for PCR, cloning the gene into a TOPO vector, transforming E. coli cells, screening clones by PCR, and purifying the expressed protein using nickel affinity chromatography and SDS-PAGE.
Nucleic acid extraction from FFPE cell blocks Caroline Seiler
This document compares several commercially available methods for extracting nucleic acids from formalin-fixed paraffin-embedded cancer cell line samples. It evaluates the yields and quality of extracted DNA and RNA using kits from Qiagen, Life Technologies, and Promega. The Qiagen RNeasy FFPE kit yielded the highest RNA amounts, while the Life Technologies PicoPure DNA kit extracted the most DNA. A miniaturized qPCR assay was developed to quantify and assess DNA quality. The document concludes that careful consideration of the extraction kit is needed depending on whether high yield or quality is prioritized for downstream applications like sequencing.
Streamlined next generation sequencing assay development using a highly multi...Thermo Fisher Scientific
Next generation sequencing (NGS) assay development for solid tumor sequencing requires characterization of variant calling directly from formalin-fixed paraffin embedded (FFPE) tissue samples. However, cell line based FFPE and human FFPE samples only contain 2 to 20 variants, which require laboratories to invest significant resources in sample sourcing and preparation when developing assays to detect 100+ variants
Cancer therapies that target specific pathways can be more effective than established, nonspecific chemotherapy and radiation treatments, and may prevent side effects on healthy tissues. Such targeted therapies can only be applied after underlying gene mutations have been identified. However, detecting low frequency variants from clinically relevant samples poses significant challenges. Specimens are routinely formalin-fixed and paraffin-embedded (FFPE) for histology, which can decrease the efficiency of NGS library preparation. In this presentation, we discuss approaches for extraction of DNA from FFPE samples, and recommend quality control assays to guide parameter selection for library construction and sequencing depth.
Cancer Research & the Challenges of FFPE Samples – An IntroductionQIAGEN
A cascade of complex genetic and epigenetic changes regulate tumor formation and progression. Gene expression analyses can shed light on these changes at a molecular level and identify the key genes and associated pathways involved in cancer. Often the samples used in cancer research are FFPE samples, which pose a significant challenge in terms of nucleic acid quality. The quality of nucleic acids extracted from FFPE samples depends on a number of factors, including how the samples were handled before, during and after fixation and embedding.
Dr. Vishwadeepak Tripathi describes the variability of sample purification from FFPE samples – in particular, samples to be used in cancer research. What are the challenges and solutions, and what quality control approach can ensure credible results? This webinar will focus on sample purification and the quality control of FFPE samples and compare different automated purification procedures.
MicroRNA Stability in FFPE Tissue Samples: Dependence on GC Contentkalahawer
MicroRNAs (miRNAs) are small non-coding RNAs responsible for fine-tuning of gene
expression at post-transcriptional level. The alterations in miRNA expression levels profoundly
affect human health and often lead to the development of severe diseases. Currently,
high throughput analyses, such as microarray and deep sequencing, are performed
in order to identify miRNA biomarkers, using archival patient tissue samples. MiRNAs are
more robust than longer RNAs, and resistant to extreme temperatures, pH, and formalinfixed
paraffin-embedding (FFPE) process. Here, we have compared the stability of miRNAs
in FFPE cardiac tissues using next-generation sequencing. The mode read length in FFPE
samples was 11 nucleotides (nt), while that in the matched frozen samples was 22 nt.
Although the read counts were increased 1.7-fold in FFPE samples, compared with those in
the frozen samples, the average miRNA mapping rate decreased from 32.0% to 9.4%.
These results indicate that, in addition to the fragmentation of longer RNAs, miRNAs are to
some extent degraded in FFPE tissues as well. The expression profiles of total miRNAs in
two groups were highly correlated (0.88 <r < 0.92). However, the relative read count of each
miRNA was different depending on the GC content (p<0.0001). The unequal degradation of
each miRNA affected the abundance ranking in the library, and miR-133a was shown to be
the most abundant in FFPE cardiac tissues instead of miR-1, which was predominant
before fixation. Subsequent quantitative PCR (qPCR) analyses revealed that miRNAs with
GC content of less than 40% are more degraded than GC-rich miRNAs (p<0.0001).We
showed that deep sequencing data obtained using FFPE samples cannot be directly compared
with that of fresh frozen samples. The combination of miRNA deep sequencing and
other quantitative analyses, such as qPCR, may improve the utility of archival FFPE tissue
samples.
PCR (polymerase chain reaction) is a method to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA. PCR is used to reproduce (amplify) selected sections of DNA or RNA.
pcr en temps réel et evolution biotecheDjamilaHEZIL
This document discusses the development and applications of real-time polymerase chain reaction (RT-PCR). Some key points:
- RT-PCR was developed in the 1990s and has revolutionized gene detection and expression analysis by allowing quantification during the reaction in real-time.
- It has widespread applications in medicine, including cancer diagnosis and monitoring treatment, as well as in plant pathology, forensics, and other fields by enabling sensitive detection of genes and genetic variations.
- Challenges include optimizing sampling and nucleic acid extraction methods for different sample types and developing multiplex assays and internal controls for accurate quantification. Overall, RT-PCR is a powerful and sensitive technique that has expanded biological research capabilities.
Styles of Scientific Reasoning, Scientific Practices and Argument in Science ...Elsa von Licy
The document discusses various topics related to scientific reasoning, practices, and argumentation including different styles of scientific thinking, features of scientific knowledge, and teaching and learning science. It provides examples of "crazy ideas" in science that are now accepted, examines the role of argument in science, and outlines the scientific practices and central questions of science. It also discusses developing models, planning investigations, analyzing data, and constructing explanations as key scientific practices.
Anti-philosophy rejects traditional philosophy and logic, instead embracing creativity, spirituality, and personality. It considers philosophy to be dead, kept alive artificially by analytic philosophers. The document criticizes how philosophy is currently taught and argues it has become unproductive, replacing original aims with nonsense. Anti-philosophy's goal is not to destroy philosophy but to transform its current state and avoid fundamentalism in philosophy and science.
There is no_such_thing_as_a_social_science_introElsa von Licy
This document provides an introduction and overview of the arguments made in the book "There is No Such Thing as Social Science". It begins by stating the provocative title and questioning whether the authors will take it back or qualify their position.
It then outlines three ways the term "social science" could be used - referring to a scientific spirit of inquiry, a shared scientific method, or reducibility to natural sciences. The authors argue against the latter two, methodological and substantive reductionism.
The introduction discusses how opponents may accuse the authors of being a priori or anti-reductionist, but argues that those defending social science are actually being dogmatic by insisting it must follow a scientific model. It frames the debate as being
1. SABiosciences
TM
TECHNICAL ARTICLE
Biomarker Discovery from Paraffin Embedded Samples
Successful Principles for Addressing FFPE-associated Real-time PCR Challenges
Yexun Wang Ph.D., Emi Arikawa Ph.D., Shankar Sellappan Ph.D., and Li Shen Ph.D.
SABiosciences Corporation 6951 Executive Way, Frederick, MD 21703 USA
Phone: +1 (301) 682-9200 Fax: +1 (301) 682-7300 Web: www.SABiosciences.com
Email: support@SABiosciences.com
Abstract: Traditional histopathological analysis of formalin-fixed paraffin-embedded (FFPE) tissue was capable of
providing expression status on a few targeted proteins or genes. Although fixed samples could serve as valuable sources
for biomarker identification studies, much of their value is hidden at the molecular level. Analysis of gene expression
profiles using genetic material contained within FFPE samples is further limited by the chemical modifications that
decrease the quality and availability of recovered RNA for molecular analysis. In this article, we present the successful
principles and results of the new RT2 FFPE PreAMPTM technology that combines a novel RNA extraction protocol
that exploits thermodynamic and structural characteristics of FFPE RNA with a powerful preamplification step of a
pathway-focused set of genes for real-time gene expression analysis. With this new technology, FFPE RNA can finally
serve as a powerful tool for restrospective and future gene profiling studies for biomarker identification.
Introduction
Biomarkers, as defined by the NIH Working Group, are “ a characteristic that is objectively measured and evaluated as an indicator of
normal biologic processes, pathogenic processes, or pharmacologic
responses to a therapeutic intervention” [1].
Since all biological
processes are carried out by genes at the molecular level, gene
expression profiles, as measured by RNA quantiation through realtime PCR analysis, have the potential to serve as predictors of diseased
conditions or response to therapeutic interventions.
Biomarker
discovery through gene expression analysis has been widely used in
basic and clinical research, with freshly prepared tissue or cultured
cells most often used as starting material. However, when fresh
samples are not readily available, as often the case with human tissue
samples, researchers rely on previously prepared, archived samples.
In spite of these difficulties, using formalin-fixed paraffin-embedded
(FFPE) samples in gene expression analysis studies also presents
great advantages. First, FFPE samples are available in vast amounts
and are readily accessible. It is estimated that there are over 400
million FFPE samples stored worldwide, with the number of samples
expected to increase annually [5]. Second, almost all FFPE samples
have associated pathological and clinical annotations. Although
faced with similar issues like institutional approval, sample quantity
acquisition, and sample heterogeneity, it is still much easier to
gather FFPE samples with different clinical outcomes as compared
to collecting fresh samples. This makes association and classification
studies much easier. Thirdly, applying biomarkers developed using
FFPE samples fits well with the traditional workflow of clinical
practices.
Most archived samples have been prepared in neutrally-buffered
formalin solutions and embedded in paraffin. Paraffin blocks serve
as excellent substrates for morphological and immunological studies;
unfortunately their use in molecular and genomic studies has proven
challenging. Technical obstacles hindering the exploitation of these
samples can be attributed to extensive chemical cross-linkings among
proteins, DNA, and RNA, as well as from RNA degradation. Crosslinking is primarily caused by formaldehyde fixation, which adds
mono-methylols (-CH2OH) to the amino groups of all four nucleic
acid bases and proteins, resulting in base modifications, methylene
bridging (N-CH2-N) between neighboring bases, and DNA-protein
and RNA-protein cross-linkages [2]. In addition, RNA degradation
begins with tissue anoxia during fixation and continues throughout
the storage period [3, 4]. These damages compromise data consistency
and sensitivity in molecular studies.
Contents
Introduction ........................................................................................1
Experimental Protocol .......................................................................2
Component 1: FFPE RNA Extraction .......................................2
Component 2: Reverse Transcription .....................................3
Component 3: qPCR Primer Design .........................................4
Component 4: PreAMP Signal Amplification .........................5
FFPE PCR Array Performance ..........................................................5
Application Examples .............................................................................6
Summary ...................................................................................................6
References ..............................................................................................7
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Reverse Transcription
Real-time PCR is the simplest and most accurate method to study
gene expression and validate expression biomarkers. Before RNA
can be analyzed by real-time PCR, it has to be converted to cDNA
in a reverse transcription step. The common strategy for reverse
transcription uses the MMLV reverse transcriptase after RNA is
primed with random hexamers or oligo dT primers. These two
priming strategies are equally efficient for most RNA when fresh
samples are used. However, when RNA from FFPE sample is used,
neither strategy can deliver reverse transcription efficiencies high
enough for later processes. Since RNA from FFPE samples is highly
fragmented, the intended amplicon region in PCR is most likely
disconnected from the poly(A) tail. Thus the oligo dT primer based
reverse transcription cannot reach the amplicon region, which leads
to lower PCR detection (Figure 2). For the same reason, most of
the random hexamers primed upstream of the target region cannot
convert RNA amplicons into cDNAs. It is expected that some
random hexamers primed immediately upstream of the target region
will convert RNA amplicons into cDNA, thereby detectable in PCR.
However, the very low concentration of those specific hexamers (the
concentration of one particular hexamer is only 1/4096th, or 0.02%,
of the total concentration) significantly limits priming efficiency, as
it is known that oligonucleotide annealing is highly concentrationdependent. From this discussion, it becomes obvious that using a
single gene-specific primer closely upstream of the target region
will have the highest efficiency in converting an RNA amplicon into
cDNA from FFPE samples.
A. cDNA Synthesis with Fresh RNA
AAAAA 3’
5’
Primer 4
Primer 3
Primer 2
Primer 1
B. cDNA Synthesis with FFPE RNA
AAAAA
Primer 1
Primer 3
Primer 2
Primer 4
Figure 2: A) For intact RNA from fresh samples, any of the primers (1, 2, 3, or 4) can
generate cDNAs which contain the target amplicon (in gray). B) For fragmented
RNA from FFPE samples, only primer 4 can generate cDNA which contains the target
amplicon.
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Figure 1: Expression rank order of 84 genes (Cancer PathwayFinder PCR Array) in RNA
extracted from one 20 mm section of a 5-year old normal human intestine FFPE block
using the RT2 FFPE RNA Extraction Kit. MicroRNA data is not shown.
Email support@SABiosciences.com
Web www.SABiosciences.com
4. RT2 FFPE RNA Extraction and PreAMPlification
4
To demonstrate that gene-specific primers will have a higher RT
efficiency than random hexamers for FFPE RNA, we designed
gene-specific RT primers for 89 genes present on the human Cancer
PathwayFinderTM PCR array (PAHS-033). We pooled 89 RT primers
together (0.225 uM each) and applied them to the product of the
RT reaction of RNA extracted from one 20 mm section of a five-year
old normal human spleen FFPE block, followed by RT2 PCR Array
analysis. For all the genes tested, the Ct values decreased an average
of 3.3 cycles, i.e. a ~10X increase in sensitivity (Figure 3). While
performing the analysis, we also noticed that some assays showed
non-specific amplification. This is likely due to non-specific primer
annealing events resulting from the low temperatures employed
during the RT step; other groups have reported similar problems
[6]. While this problem with non-specific amplification can be solved
through individual assay optimization, it limits the genome-wide
application of this approach for gene expression analysis on FFPE
samples. Therefore we sought alternative strategies to improve assay
sensitivity.
To test this hypothesis, we selected 22 genes which have existing RT2
primer designs with amplicon sizes longer than 130 bp. The average
size was 162 bp, with the longest at 191 bp. We then redesigned primers
for these genes so the new amplicon sizes range from 53 to 81 bp
(average = 67 bp), and confirmed the new designs work as efficiently
as the old ones. We then generated cDNA from a normal five-year old
human spleen FFPE block and compared the real-time PCR results of
these two sets of primers. As expected, the shorter amplicon designs,
in general, yielded Ct values much lower than the longer designs for
the same amount of cDNA (Figure 4A). The average Ct gain is 2.8
cycles, with the largest gain of over 5 cycles for some genes. Thus, by
simply designing shorter amplicons, we can increase PCR sensitivity
by almost 10-fold. In contrast, shorter amplicon designs do not
improve Cts for RNAs of high quality (Figure 4B).
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Random Primer
GS Primer
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Figure 3: Gene Specific RT Primer Improves Reverse Transcription Efficiency. 500
ng RNA extracted from one 20 mm section of a five-year old human spleen FFPE block
were reversed transcribed using either random hexamers or a pool of 89 gene specific
primers located upstream of the PCR amplicons for the human Cancer PathwayFinderTM
PCR Array. Equal volumes of cDNA were later used on the same PCR array. Shown here
are the box plots of raw Ct values for each condition.
The sizes of RNA fragments extracted from FFPE samples usually
range from 50 to 300 nt, with most around 100 nt. This average size
correlates well with the age of the block, with older blocks yielding
shorter RNA fragments [4]. This indicates RNA fragmentation
continues even after the sample is completely fixed and embedded.
These short RNA fragments necessitate specialized PCR primer
designs, with an important caveat being the PCR amplicon should
not be much longer than the average size of the RNA fragments.
This stipulation can be explained by recognizing that there is less
probability to amplify a sequence fragment with a size much longer
than the average size of all RNA fragments. For example, when initial
RNA samples are randomly fragmented, the percentage of 300 nt
long fragments is much lower than that of 100 nt long fragments,
when the average size of fragments is 100 nt. It can be inferred that
shorter amplicon designs should yield better sensitivity in real-time
PCR analysis for FFPE samples.
Tel 888-503-3187 (USA)
301-682-9200
Short Amplicon
Regular Amplicon
Short Amplicon
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qPCR Primer Design
Regular Amplicon
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Figure 4: Primers Designed for Shorter PCR Amplicons Generally Yield Lower Cts for
Fragmented RNA. A) 500 ng RNA extracted from one 20 mm section of a five-year old
human spleen block was reversed transcribed using random hexamers. Equal amounts
of cDNA were used on two customized RT2 PCR Arrays. For one array, primers were
designed to generate regular PCR amplicons (132 bp to 191 bp). For the second array,
primers were designed to specifically generate shorter amplicons (53 to 81 bp) for the
same genes. Shown here are the before and after raw Ct values for each gene. B) cDNA
transcribed from 1 ug high quality human universal RNA was used on 45 genes (from
PAHS-060) with two different primer designs. For one array, primers were designed to
generate regular PCR amplicons (130 bp to 195 bp). For the second array, primers were
designed to specifically generate shorter amplicons (50 to 89 bp) for the same genes.
Shown here are the before and after raw Ct values for each gene.
Fax 888-465-9859 (USA)
301-682-7300
5. SABiosciences
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PreAMP Signal Amplification
Another discouraging factor for FFPE samples is that while one can
extract RNA in the microgram range, due to damages, the amount of
RNA fragments which can serve as effective templates in RT-qPCR is
much less. A pre-amplification approach has been used to enhance
detection of RNA extracted from small fresh samples (e.g. from LCM,
FNB, cell sorting), and it is expected that pre-amplification can also be
used to boost the overall qPCR assay sensitivity in FFPE samples.
FFPE PCR Array Performance
In order to demonstrate the value of pre-amplification when working
with FFPE samples yielding low effective RNA template, we have
developed the RT2 FFPE PreAMP technology. When used together
with the RT2 PCR array platform, multi-gene expression analysis on
FFPE samples can be successfully performed. After RNA extracted
from FFPE samples is first converted to cDNA, a quarter of the cDNA
is used in a tightly-controlled multiplex PCR reaction comprised
of a mixture of primers specific to those genes on a particular PCR
Array. After the PreAMP reaction is complete and excess primers are
removed by enzyme digestion, the pre-amplified cDNA is distributed
across a PCR array for individual gene detection.
Compared to the previously discussed strategies for RT-qPCR primer
design, the FFPE PreAMP technology yields the best improvement in
sensitivity. Depending on the cycling numbers of the multiplex PCR,
qPCR assay sensitivity can be easily improved by at least 100-fold or
more for FFPE RNA. As an example, we extracted RNA from a fiveyear old normal human intestine FFPE block, converted it into cDNA,
and ran the cDNA on the RT2 Human Cancer PathwayFinder PCR
Array with or without the FFPE PreAMP technology. The results
from our regular PCR array protocol without FFPE PreAMPlification
showed 18 genes (20% of the array) as “absent” due to Ct values
greater than 35. On the other hand, addition of the FFPE PreAMP
technology greatly improved the detection of those 18 genes (Figure
5), increasing the positive call rate to 100%.
Email support@SABiosciences.com
RT
RT + PreAMP
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BAD
NME4
TEK
JUN
COL18A1
SERPINB5
TNFRSF1A
TNFRSF25
CDC25A
TP53
ANGPT1
BRCA1
MET
TERT
TIMP3
TWIST1
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CASP8
When scientists try to look at the expression profile of multiple genes
on the same sample by qPCR, the easiest approach is to run multiple
individual qPCR assays corresponding to those genes. This creates
the problem of sample division or dilution. For example, if one tries
to use qPCR to study 100 genes including gene A using 1 mg of RNA,
he would have to divide 1 mg RNA into 100 assays, one assay for each
gene. If gene A only has 100 copies in 1 mg RNA, then its copy number
in the individual assay designated for gene A will drop down to one,
which cannot be reliably detected. From another perspective, the
reverse transcription reaction usually has to be diluted before cDNA
can be used in qPCR due to the interference of RT chemistry with
qPCR chemistry. This also causes the number of original gene copies
to be diluted and fall off the qPCR detection range. This problem
can be avoided by starting with much more RNA in the experiment.
However, this is not always feasible, especially with FFPE samples.
Figure 5: PreAMPTM Process Makes More Genes Detectable in FFPE Samples. RNA
extracted from a five-year old human intestine FFPE block was reverse transcribed with
or without the PreAMP process. Both cDNAs were run on Human Cancer PathwayFinder
arrays (PAHS-033). Shown here are 18 genes which are called “Absent” using standard
RT-PCR array procedures.
Any signal or sample amplification approach needs to have high
fidelity in order to maintain the original profile of the genes being
studied. We evaluated the consistency of the PreAMP technology and
the fidelity of gene expression profiles between the original sample
and samples undergoing PreAMPlification. The PreAMPlified
samples showed very high consistency between two independent
runs (Figure 6).
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Figure 6: Inter-assay Consistency of the PreAMP Process. Two independent PreAMP
reactions were set up using the same amount of mouse cDNA. Mouse Toll-Like
Receptor Signaling Pathway PCR Array (PAMM-018) was used to evaluate the inter-run
consistency. DCt (Ct(GOI) – Ct(HKG)) of 89 genes from two separate runs shows R > 0.99.
Web www.SABiosciences.com
6. RT2 FFPE RNA Extraction and PreAMPlification
6
Application Examples
To evaluate the biological significance of the fold changes, we used
RNAs from human spleen and intestine FFPE blocks and examined
the differential expression of 89 genes in the Human Cancer
PathwayFinder PCR Array. We compared the results obtained using
our PCR Array protocol with those incorporating the FFPE PreAMP
protocol. When looking at those genes which are reliably detected as
“present” (Ct < 31.5) with the PCR array, their raw Ct values without
PreAMP are much higher, but highly correlated with the raw Ct
values after the PreAMP process (Figure 7).
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FFPE PreAMP Ct
The fold changes, or the difference in gene expression between
spleen and intestine samples, also correlate well between the two
protocols (Figure 8). This suggests two conclusions that establish
full confidence in providing biologically relevant and accurate data.
First, the original gene expression profile is faithfully maintained
during the PreAMP process. In a population of genes with both high
and low expressers, preamplification amplifies all genes equally such
that overall sensitivity of detection increases, while the differential
patterning is maintained. Second, the PreAMP technology allows one
to compare multiple genes simultaneously, some of which cannot be
detected by traditional means.
R = 0.96
R = 0.98
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Figure 8: Comparable Fold Change Results Between PreAMP and Unamplified FFPE
Samples. RNA extracted from human spleen and intestine FFPE blocks were reverse
transcribed with or without the PreAMP process. All four cDNAs were run on PAHS033. Shown here is the DDCt comparison. All the genes show significantly changed in
original FFPE samples were also detected as significantly changed in the same direction
after the PreAMP process (in white regions A and B). Although some genes showed
fold change values (as identified by the arrow) in the opposite directions, the values
were not significant in either condition. Only genes which have Cts lower than 31.5 in
both unamplified spleen and intestine samples are shown here.
40
No PreAMP
PreAMP
30
20
Summary
10
0
5
10
15
20
25
30
35
40
45
50
Gene Assays in Ct Ranking Order
Figure 7: Highly comparable Ct values between PreAMP and non-PreAMP FFPE
Spleen RNA. RNA extracted from human spleen FFPE block was reverse transcribed
with or without the PreAMP process. Both cDNAs were run on the Human Cancer
PathwayFinder Array. A) Shown here is the scatter plot of raw Cts. B) Every assay is
improved by approximately the same number of Cts after the PreAMP process. Each
pair of data points (PreAMP and no PreAMP) represent a different gene. Only genes
which have Cts lower than 31.5 in the reaction without PreAMP are shown here.
Tel 888-503-3187 (USA)
301-682-9200
The RT2 FFPE PreAMP technology solves the major challenge in realtime PCR based gene expression analysis using FFPE samples. It
greatly improves the detection limit of FFPE RNA. It also allows for
easy integration with the popular RT2 Profiler PCR Array platform
for analysis of a pathway-focused set of genes.
As millions of FFPE samples are currently stored in archives with
thorough pathological information and clinical history of the patient
available, using them as a source for biomarker discovery has gained
increasing importance. With the improvement in assay design
and the introduction of a novel workflow tailored to the nature of
these samples, we can now expect to have better utilization of these
valuable resources. We believe and demonstrate that our complete
FFPE RNA extraction protocol, FFPE PreAMP technology and PCR
Array system will be a valuable tool in this quest.
Fax 888-465-9859 (USA)
301-682-7300
7. SABiosciences
7
References
1. Biomarkers and surrogate endpoints: preferred definitions and
conceptual framework. Biomarkers Definitions Working Group. Clin
Pharmacol Ther. 2001 Mar;69(3):89-95.
2. Analysis of chemical modification of RNA from formalin-fixed
samples and optimization of molecular biology applications for such
samples. Masuda N, Ohnishi T, Kawamoto S, Monden M, Okubo K.
Nucleic Acids Res. 1999 Nov 15;27(22):4436-43.
3. Determinants of RNA quality from FFPE samples. von Ahlfen
S, Missel A, Bendrat K, Schlumpberger M. PLoS One. 2007 Dec
5;2(12):e1261.
4. Measurement of gene expression in archival paraffin-embedded
tissues: development and performance of a 92-gene reverse
transcriptase-polymerase chain reaction assay. Cronin M, Pho M,
Dutta D, Stephans JC, Shak S, Kiefer MC, Esteban JM, Baker JB. Am J
Pathol. 2004 Jan;164(1):35-42.
5. Applying aCGH to molecular diagnostics. Kathy Liszewski. GEN
2008 May 15; 28(10).
6. Enhanced detection of RNA from paraffin-embedded tissue using
a panel of truncated gene-specific primers for reverse transcription.
Mikhitarian K, Reott S, Hoover L, Allen A, Cole DJ, Gillanders WE,
Mitas M. Biotechniques. 2004 Mar;36(3):474-8.
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