This document discusses factors that impact gene expression analysis and describes various amplification consumables from Bio-Rad Laboratories. It covers RNA isolation kits that purify RNA from different sample types and ensure DNA removal. It also discusses reagents for reverse transcription and real-time qPCR that provide accurate cDNA synthesis and sensitive detection. Finally, it mentions PCR plastic consumables that are compatible with instrumentation and avoid contamination.
GenTegra RNA Overview - Short & long term stabilizationkbebak
GenTegra RNA is a product that stabilizes RNA samples at ambient temperature for long-term storage, transport, and handling. According to ongoing experiments, RNA samples preserved with GenTegra RNA showed no degradation after 3.5 years of mixed temperature dry state storage at ambient temperature. After rehydration, the samples performed identically to frozen controls in downstream applications. GenTegra RNA protects RNA samples by stopping RNase activity in solution and preventing hydrolysis and oxidation when dried, allowing indefinite storage at ambient temperature.
Some sample sources present special challenges in RNA isolation or contain substances that cause problems in RNA analysis. These guides to RNA isolation have tips for a whole range of sample types, including guidance on the best kits for each.
Improving RNA yield and quality is easy with the right protocols. This trouble shooting guide presents methods to overcome common difficulties in RNA purification. Prevent RNA degradation, improve RNA yields, reduce genomic DNA contamination, and more.
olymerase chain reaction (PCR) is a method widely used in molecular biology to make several copies of a specific DNA segment. Using PCR, copies of DNA sequences are exponentially amplified to generate thousands to millions of more copies of that particular DNA segment.
The document describes a collection of lentiviral ORF clones called Precision LentiORFs contained in the pLOC lentiviral vector. Key features include:
- The ORF, fluorescent marker, and selection marker are expressed from one promoter for tracking ORF expression.
- It provides two methods (fluorescent marker and antibiotic resistance) to optimize transduction.
- The vector allows addition of fusion tags and works with both transfection and transduction for ORF expression in a variety of cell types.
- Protocols are provided for replicating plates of clones stored in glycerol stock and preparing plasmid DNA from individual clones.
The document discusses the challenge of identifying effector genes in the wheat stripe rust fungus Puccinia striiformis f.sp. tritici. Effector genes are small secreted proteins that help the fungus infect wheat plants. Next-generation sequencing allows genomic and transcriptomic analysis but has limitations in assembling repetitive sequences like effectors. The author has analyzed transcriptomes of the fungus grown in planta to predict 100 small secreted protein candidates as potential effector genes for further laboratory tests. Identifying the fungus's effector genes could help develop resistant wheat varieties to reduce annual losses from stripe rust in Australian wheat production.
The document discusses nucleic acid amplification techniques, specifically polymerase chain reaction (PCR). It explains that PCR involves denaturing DNA, annealing primers, and extending DNA strands through repeated heating and cooling cycles to exponentially amplify a specific DNA target. Key aspects of PCR covered include its invention by Kary Mullis, use of Taq polymerase, and applications such as disease diagnosis using techniques like real-time PCR.
Rapid extraction of high yield, high quality DNA from tissue samples - Downlo...QIAGEN
Genetic and genomic analysis from tissue samples requires the extraction of high quality DNA. Mechanical disruption methods such as bead milling provide high yield from tissue samples, but cause damage to the nucleic acids. Purely enzymatic methods such as proteinase K digestion can extract nucleic acid without damage, but require long incubation times, often proceeding overnight, and without approaching the yields achieved by mechanical disruption techniques. Thus a method is needed which can provide a rapid extraction of high yield, high quality DNA from tissue samples. See the new method.
GenTegra RNA Overview - Short & long term stabilizationkbebak
GenTegra RNA is a product that stabilizes RNA samples at ambient temperature for long-term storage, transport, and handling. According to ongoing experiments, RNA samples preserved with GenTegra RNA showed no degradation after 3.5 years of mixed temperature dry state storage at ambient temperature. After rehydration, the samples performed identically to frozen controls in downstream applications. GenTegra RNA protects RNA samples by stopping RNase activity in solution and preventing hydrolysis and oxidation when dried, allowing indefinite storage at ambient temperature.
Some sample sources present special challenges in RNA isolation or contain substances that cause problems in RNA analysis. These guides to RNA isolation have tips for a whole range of sample types, including guidance on the best kits for each.
Improving RNA yield and quality is easy with the right protocols. This trouble shooting guide presents methods to overcome common difficulties in RNA purification. Prevent RNA degradation, improve RNA yields, reduce genomic DNA contamination, and more.
olymerase chain reaction (PCR) is a method widely used in molecular biology to make several copies of a specific DNA segment. Using PCR, copies of DNA sequences are exponentially amplified to generate thousands to millions of more copies of that particular DNA segment.
The document describes a collection of lentiviral ORF clones called Precision LentiORFs contained in the pLOC lentiviral vector. Key features include:
- The ORF, fluorescent marker, and selection marker are expressed from one promoter for tracking ORF expression.
- It provides two methods (fluorescent marker and antibiotic resistance) to optimize transduction.
- The vector allows addition of fusion tags and works with both transfection and transduction for ORF expression in a variety of cell types.
- Protocols are provided for replicating plates of clones stored in glycerol stock and preparing plasmid DNA from individual clones.
The document discusses the challenge of identifying effector genes in the wheat stripe rust fungus Puccinia striiformis f.sp. tritici. Effector genes are small secreted proteins that help the fungus infect wheat plants. Next-generation sequencing allows genomic and transcriptomic analysis but has limitations in assembling repetitive sequences like effectors. The author has analyzed transcriptomes of the fungus grown in planta to predict 100 small secreted protein candidates as potential effector genes for further laboratory tests. Identifying the fungus's effector genes could help develop resistant wheat varieties to reduce annual losses from stripe rust in Australian wheat production.
The document discusses nucleic acid amplification techniques, specifically polymerase chain reaction (PCR). It explains that PCR involves denaturing DNA, annealing primers, and extending DNA strands through repeated heating and cooling cycles to exponentially amplify a specific DNA target. Key aspects of PCR covered include its invention by Kary Mullis, use of Taq polymerase, and applications such as disease diagnosis using techniques like real-time PCR.
Rapid extraction of high yield, high quality DNA from tissue samples - Downlo...QIAGEN
Genetic and genomic analysis from tissue samples requires the extraction of high quality DNA. Mechanical disruption methods such as bead milling provide high yield from tissue samples, but cause damage to the nucleic acids. Purely enzymatic methods such as proteinase K digestion can extract nucleic acid without damage, but require long incubation times, often proceeding overnight, and without approaching the yields achieved by mechanical disruption techniques. Thus a method is needed which can provide a rapid extraction of high yield, high quality DNA from tissue samples. See the new method.
The 10 basic tips & tricks presented in this slide-deck are based on Frequently Asked Questions raised by scientists, and their answers as supplied by the Ambion technical support teams at Life Technologies.
Micro RNARNA INTERFERENCE AND ITS APPLICATIONS IN CROP IMPROVE...SANIVARAPUNAGALAKSHM
This document provides an overview of RNA interference (RNAi) and its applications in crop improvement. It discusses the history and discovery of RNAi, the mechanism of RNAi involving initiation by Dicer and effector function of RISC complexes, and methods of transforming plants with RNAi constructs. Applications of RNAi described in the document include modifying traits in rice, maize, barley, cotton, jute, tomato, lathyris, and coffee to improve nutritional quality, increase yields, confer virus resistance, and remove toxic compounds.
This document describes Norgen Biotek Corporation, a company that provides sample preparation kits for RNA, microRNA, DNA and protein purification from various sample types. It offers over 40 kits for total RNA isolation without the use of phenol, including kits for cultured cells, tissues, blood, and formalin-fixed paraffin-embedded samples. The document highlights that Norgen's kits recover a full size range of RNA, including small RNA and microRNA, and provide high quality RNA suitable for various downstream applications. It also provides contact information, ordering details, and an overview of Norgen's RNA purification kits and their applications.
Back to basics: Fundamental Concepts and Special Considerations in RNA IsolationQIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the challenges and considerations of handling RNA samples, RNA stabilization, the need for quality control analysis and common methods for RNA integrity and quality assessment.
This document discusses various molecular biology concepts and techniques used in DNA and protein analysis. It defines key terms like DNA, RNA, genes, transcription, translation and genome. It also describes commonly used techniques like PCR, gel electrophoresis, Southern blotting and different types of genetic markers. The document provides details on the basic principles, applications and differences between techniques like RFLP, AFLP, RAPD analysis.
This document discusses molecular biology techniques. It covers topics like DNA, RNA, proteins, transcription, translation, genome analysis, and basic tools used in DNA techniques like PCR and electrophoresis. PCR allows for copying and altering DNA sequences. Primers are short DNA strands that serve as starting points for DNA synthesis during PCR. Overall, the document provides an overview of key concepts and techniques in molecular biology.
Tools and Techniques (Molecular & Biochemical) to Study Physiological Process...Agronomist Wasim
The document discusses various molecular and biochemical tools and techniques used to study physiological processes and stress responses in plants, including DNA and RNA isolation, cDNA synthesis and library construction, RT-PCR, Northern blot, and immunoassays. It provides details on how to perform techniques like DNA isolation, RNA isolation from yeast, cDNA library construction, and the different types and applications of RT-PCR, Northern blot, and categories of immunoassays.
This document describes Norgen Biotek's Plant/Fungi Total RNA Purification Kit, which provides a rapid method for isolating total RNA, including microRNA, from plant and fungal samples. The kit uses a spin column format to purify RNA from fresh or frozen tissues without phenol/chloroform extractions. It isolates all sizes of RNA, from mRNA to microRNA. The purified RNA is of high quality and quantity and can be used in downstream applications such as PCR and expression arrays. Norgen also offers the kit in a 96-well format for high-throughput purification of plant/fungi RNA.
Technical Guide to Qiagen PCR Arrays - Download the GuideQIAGEN
Total RNA discovery with RT2 and miScript PCR Arrays : Explore the RNA universe - Whatever your destination within the RNA universe, QIAGEN will help you get there. The miRNeasy kits deliver pure, high-quality total RNA from a broad range of samples. The RT2 and miScript PCR arrays are a complete solution both for focused analysis of gene and microRNA expression and for validation of microarray and RNA sequencing experiments. Together with the powerful analytics tools of GeneGlobe® and QIAGEN Ingenuity® Pathway Analysis, these products give you a smooth path from your sample to high-quality results.
1. mRNA isolation is the process of extracting messenger RNA from biological samples. It is important for research and industry applications as mRNA provides insight into which genes are being expressed and translated into proteins.
2. Total RNA is first extracted using Trizol, which separates RNA, DNA and proteins. mRNA is then isolated from total RNA using biotin-labeled oligo dT probes that selectively bind to the poly-A tail of mRNA molecules.
3. The mRNA-probe complexes are immobilized on magnetic beads coated with streptavidin. This allows the mRNA to be separated and purified from other RNAs through magnetic separation washes. The purified mRNA can then be used in applications like RT-PCR and protein
This document outlines the workflow and methods for an RNA lab involving isolation of RNA from Arabidopsis leaves, quantification of the RNA, reverse transcription to cDNA, and quantitative PCR (qPCR) analysis. The workflow takes place over two days, with RNA isolation and quantification on day 1 and setting up and analyzing qPCR on day 2. Students will be evaluated through an in-class quiz worth 50 points and a lab report worth 50 points. The document provides details on the RNA isolation, quantification and quality check methods, as well as background information and considerations for the qPCR analysis.
Take your RNA research to the next level with QIAGEN LNA tools!QIAGEN
Download the flyer!
Experience truly exceptional RNA research with QIAGEN's next-generation, LNA®-enhanced tools. LNA (Locked Nucleic Acid) oligos bind with much higher affinity and specificity to RNA targets than standard DNA and RNA oligos – This enables specific and sensitive detection of small RNAs and discrimination between highly similar
sequences.
QIAGEN provides kits for purifying RNA from a variety of sources including animal cells, tissues, bacteria, yeast, and plants. The RNeasy Mini kits use silica membrane technology to selectively bind and purify RNA over 200 nucleotides in length. Samples are lysed in a guanidine-based buffer to immediately inactivate RNases, followed by binding of RNA to the RNeasy membrane. Contaminants are washed away and high-quality RNA is eluted for downstream applications such as RT-PCR. Protocols are included for different starting materials including cells, tissues, bacteria, yeast, and plants.
RNAi (RNA interference/ Gene Silencing) and its importanceKaurKawaljeet
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is a conserved biological response to double-stranded RNA that mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes. Small pieces of RNA can shut down protein translation by binding to the messenger RNAs that code for those proteins. RNA interference is already proving to be an invaluable research tool, allowing much more rapid characterization of the function of known genes. More importantly, the technology considerably bolsters functional genomics to aid in the identification of novel genes involved in disease processes.
RNA isolation is the purification of RNA from biological samples. It requires strict precautions to avoid degradation by RNases, enzymes that naturally degrade RNA. The TRIzol method uses phenol and chloroform to separate RNA, DNA and proteins during cell lysis and homogenization. Chloroform separates the mixture into aqueous and organic phases, with RNA remaining in the aqueous phase. RNA is then precipitated with isopropanol and washed with ethanol before being re-dissolved in water. RNA concentration and purity is determined spectroscopically. Filter-based and magnetic particle methods provide alternative RNA isolation techniques.
Role of Antisense and RNAi-based Gene Silencing in Crop ImprovementMariya Zaman
This document presents information on RNA interference (RNAi) and its application in crop improvement. It discusses the discovery of antisense RNA and RNAi technology. The mechanisms of antisense technology and RNAi are described. Advantages of RNAi include its ability to study essential genes and its high specificity. Applications include crop protection and gene therapy. Case studies demonstrate improved insect resistance in transgenic tobacco plants and the role of miRNAs in syncytium formation induced by cyst nematodes.
RNA processing is required to generate mature RNA molecules from primary transcripts. It involves 5' capping, 3' cleavage and polyadenylation, and splicing. During 5' capping, a 5' cap structure is added to primary transcripts. 3' cleavage and polyadenylation generates the mature 3' end through endonucleolytic cleavage followed by poly(A) tail addition. Splicing removes introns from primary transcripts in eukaryotes, joining exons to form mature mRNAs.
This document summarizes a seminar presentation on antisense RNA technology. The presentation covered:
1. The introduction defined antisense RNA and its potential for crop improvement to meet rising global food demand.
2. The mechanisms of antisense RNA technology were explained, including how antisense RNA binds to mRNA to inhibit translation and activate RNase H degradation.
3. The history of antisense technology was discussed, including its first observation in nature's HOK/SOK system and early experiments in the 1990s that helped define gene silencing.
Antisense RNA technology & its role in crop improvement ppt surendra singhDrSurendraSingh2
This document discusses antisense RNA technology and its role in crop improvement. It begins by introducing antisense RNA as a method for inhibiting gene expression through complementary base pairing. It then discusses various applications of antisense RNA technology in crop improvement, including delaying fruit ripening in tomato and flower senescence in carnation, producing male sterility in petunia, and reducing neurotoxins in crops like khesari. The document concludes by noting that antisense RNA technology is an efficient gene knockdown method that could be useful for genetic improvement in many plant species.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
The 10 basic tips & tricks presented in this slide-deck are based on Frequently Asked Questions raised by scientists, and their answers as supplied by the Ambion technical support teams at Life Technologies.
Micro RNARNA INTERFERENCE AND ITS APPLICATIONS IN CROP IMPROVE...SANIVARAPUNAGALAKSHM
This document provides an overview of RNA interference (RNAi) and its applications in crop improvement. It discusses the history and discovery of RNAi, the mechanism of RNAi involving initiation by Dicer and effector function of RISC complexes, and methods of transforming plants with RNAi constructs. Applications of RNAi described in the document include modifying traits in rice, maize, barley, cotton, jute, tomato, lathyris, and coffee to improve nutritional quality, increase yields, confer virus resistance, and remove toxic compounds.
This document describes Norgen Biotek Corporation, a company that provides sample preparation kits for RNA, microRNA, DNA and protein purification from various sample types. It offers over 40 kits for total RNA isolation without the use of phenol, including kits for cultured cells, tissues, blood, and formalin-fixed paraffin-embedded samples. The document highlights that Norgen's kits recover a full size range of RNA, including small RNA and microRNA, and provide high quality RNA suitable for various downstream applications. It also provides contact information, ordering details, and an overview of Norgen's RNA purification kits and their applications.
Back to basics: Fundamental Concepts and Special Considerations in RNA IsolationQIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the challenges and considerations of handling RNA samples, RNA stabilization, the need for quality control analysis and common methods for RNA integrity and quality assessment.
This document discusses various molecular biology concepts and techniques used in DNA and protein analysis. It defines key terms like DNA, RNA, genes, transcription, translation and genome. It also describes commonly used techniques like PCR, gel electrophoresis, Southern blotting and different types of genetic markers. The document provides details on the basic principles, applications and differences between techniques like RFLP, AFLP, RAPD analysis.
This document discusses molecular biology techniques. It covers topics like DNA, RNA, proteins, transcription, translation, genome analysis, and basic tools used in DNA techniques like PCR and electrophoresis. PCR allows for copying and altering DNA sequences. Primers are short DNA strands that serve as starting points for DNA synthesis during PCR. Overall, the document provides an overview of key concepts and techniques in molecular biology.
Tools and Techniques (Molecular & Biochemical) to Study Physiological Process...Agronomist Wasim
The document discusses various molecular and biochemical tools and techniques used to study physiological processes and stress responses in plants, including DNA and RNA isolation, cDNA synthesis and library construction, RT-PCR, Northern blot, and immunoassays. It provides details on how to perform techniques like DNA isolation, RNA isolation from yeast, cDNA library construction, and the different types and applications of RT-PCR, Northern blot, and categories of immunoassays.
This document describes Norgen Biotek's Plant/Fungi Total RNA Purification Kit, which provides a rapid method for isolating total RNA, including microRNA, from plant and fungal samples. The kit uses a spin column format to purify RNA from fresh or frozen tissues without phenol/chloroform extractions. It isolates all sizes of RNA, from mRNA to microRNA. The purified RNA is of high quality and quantity and can be used in downstream applications such as PCR and expression arrays. Norgen also offers the kit in a 96-well format for high-throughput purification of plant/fungi RNA.
Technical Guide to Qiagen PCR Arrays - Download the GuideQIAGEN
Total RNA discovery with RT2 and miScript PCR Arrays : Explore the RNA universe - Whatever your destination within the RNA universe, QIAGEN will help you get there. The miRNeasy kits deliver pure, high-quality total RNA from a broad range of samples. The RT2 and miScript PCR arrays are a complete solution both for focused analysis of gene and microRNA expression and for validation of microarray and RNA sequencing experiments. Together with the powerful analytics tools of GeneGlobe® and QIAGEN Ingenuity® Pathway Analysis, these products give you a smooth path from your sample to high-quality results.
1. mRNA isolation is the process of extracting messenger RNA from biological samples. It is important for research and industry applications as mRNA provides insight into which genes are being expressed and translated into proteins.
2. Total RNA is first extracted using Trizol, which separates RNA, DNA and proteins. mRNA is then isolated from total RNA using biotin-labeled oligo dT probes that selectively bind to the poly-A tail of mRNA molecules.
3. The mRNA-probe complexes are immobilized on magnetic beads coated with streptavidin. This allows the mRNA to be separated and purified from other RNAs through magnetic separation washes. The purified mRNA can then be used in applications like RT-PCR and protein
This document outlines the workflow and methods for an RNA lab involving isolation of RNA from Arabidopsis leaves, quantification of the RNA, reverse transcription to cDNA, and quantitative PCR (qPCR) analysis. The workflow takes place over two days, with RNA isolation and quantification on day 1 and setting up and analyzing qPCR on day 2. Students will be evaluated through an in-class quiz worth 50 points and a lab report worth 50 points. The document provides details on the RNA isolation, quantification and quality check methods, as well as background information and considerations for the qPCR analysis.
Take your RNA research to the next level with QIAGEN LNA tools!QIAGEN
Download the flyer!
Experience truly exceptional RNA research with QIAGEN's next-generation, LNA®-enhanced tools. LNA (Locked Nucleic Acid) oligos bind with much higher affinity and specificity to RNA targets than standard DNA and RNA oligos – This enables specific and sensitive detection of small RNAs and discrimination between highly similar
sequences.
QIAGEN provides kits for purifying RNA from a variety of sources including animal cells, tissues, bacteria, yeast, and plants. The RNeasy Mini kits use silica membrane technology to selectively bind and purify RNA over 200 nucleotides in length. Samples are lysed in a guanidine-based buffer to immediately inactivate RNases, followed by binding of RNA to the RNeasy membrane. Contaminants are washed away and high-quality RNA is eluted for downstream applications such as RT-PCR. Protocols are included for different starting materials including cells, tissues, bacteria, yeast, and plants.
RNAi (RNA interference/ Gene Silencing) and its importanceKaurKawaljeet
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is a conserved biological response to double-stranded RNA that mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes. Small pieces of RNA can shut down protein translation by binding to the messenger RNAs that code for those proteins. RNA interference is already proving to be an invaluable research tool, allowing much more rapid characterization of the function of known genes. More importantly, the technology considerably bolsters functional genomics to aid in the identification of novel genes involved in disease processes.
RNA isolation is the purification of RNA from biological samples. It requires strict precautions to avoid degradation by RNases, enzymes that naturally degrade RNA. The TRIzol method uses phenol and chloroform to separate RNA, DNA and proteins during cell lysis and homogenization. Chloroform separates the mixture into aqueous and organic phases, with RNA remaining in the aqueous phase. RNA is then precipitated with isopropanol and washed with ethanol before being re-dissolved in water. RNA concentration and purity is determined spectroscopically. Filter-based and magnetic particle methods provide alternative RNA isolation techniques.
Role of Antisense and RNAi-based Gene Silencing in Crop ImprovementMariya Zaman
This document presents information on RNA interference (RNAi) and its application in crop improvement. It discusses the discovery of antisense RNA and RNAi technology. The mechanisms of antisense technology and RNAi are described. Advantages of RNAi include its ability to study essential genes and its high specificity. Applications include crop protection and gene therapy. Case studies demonstrate improved insect resistance in transgenic tobacco plants and the role of miRNAs in syncytium formation induced by cyst nematodes.
RNA processing is required to generate mature RNA molecules from primary transcripts. It involves 5' capping, 3' cleavage and polyadenylation, and splicing. During 5' capping, a 5' cap structure is added to primary transcripts. 3' cleavage and polyadenylation generates the mature 3' end through endonucleolytic cleavage followed by poly(A) tail addition. Splicing removes introns from primary transcripts in eukaryotes, joining exons to form mature mRNAs.
This document summarizes a seminar presentation on antisense RNA technology. The presentation covered:
1. The introduction defined antisense RNA and its potential for crop improvement to meet rising global food demand.
2. The mechanisms of antisense RNA technology were explained, including how antisense RNA binds to mRNA to inhibit translation and activate RNase H degradation.
3. The history of antisense technology was discussed, including its first observation in nature's HOK/SOK system and early experiments in the 1990s that helped define gene silencing.
Antisense RNA technology & its role in crop improvement ppt surendra singhDrSurendraSingh2
This document discusses antisense RNA technology and its role in crop improvement. It begins by introducing antisense RNA as a method for inhibiting gene expression through complementary base pairing. It then discusses various applications of antisense RNA technology in crop improvement, including delaying fruit ripening in tomato and flower senescence in carnation, producing male sterility in petunia, and reducing neurotoxins in crops like khesari. The document concludes by noting that antisense RNA technology is an efficient gene knockdown method that could be useful for genetic improvement in many plant species.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
leewayhertz.com-AI in predictive maintenance Use cases technologies benefits ...alexjohnson7307
Predictive maintenance is a proactive approach that anticipates equipment failures before they happen. At the forefront of this innovative strategy is Artificial Intelligence (AI), which brings unprecedented precision and efficiency. AI in predictive maintenance is transforming industries by reducing downtime, minimizing costs, and enhancing productivity.
Taking AI to the Next Level in Manufacturing.pdfssuserfac0301
Read Taking AI to the Next Level in Manufacturing to gain insights on AI adoption in the manufacturing industry, such as:
1. How quickly AI is being implemented in manufacturing.
2. Which barriers stand in the way of AI adoption.
3. How data quality and governance form the backbone of AI.
4. Organizational processes and structures that may inhibit effective AI adoption.
6. Ideas and approaches to help build your organization's AI strategy.
Salesforce Integration for Bonterra Impact Management (fka Social Solutions A...Jeffrey Haguewood
Sidekick Solutions uses Bonterra Impact Management (fka Social Solutions Apricot) and automation solutions to integrate data for business workflows.
We believe integration and automation are essential to user experience and the promise of efficient work through technology. Automation is the critical ingredient to realizing that full vision. We develop integration products and services for Bonterra Case Management software to support the deployment of automations for a variety of use cases.
This video focuses on integration of Salesforce with Bonterra Impact Management.
Interested in deploying an integration with Salesforce for Bonterra Impact Management? Contact us at sales@sidekicksolutionsllc.com to discuss next steps.
Letter and Document Automation for Bonterra Impact Management (fka Social Sol...Jeffrey Haguewood
Sidekick Solutions uses Bonterra Impact Management (fka Social Solutions Apricot) and automation solutions to integrate data for business workflows.
We believe integration and automation are essential to user experience and the promise of efficient work through technology. Automation is the critical ingredient to realizing that full vision. We develop integration products and services for Bonterra Case Management software to support the deployment of automations for a variety of use cases.
This video focuses on automated letter generation for Bonterra Impact Management using Google Workspace or Microsoft 365.
Interested in deploying letter generation automations for Bonterra Impact Management? Contact us at sales@sidekicksolutionsllc.com to discuss next steps.
Skybuffer SAM4U tool for SAP license adoptionTatiana Kojar
Manage and optimize your license adoption and consumption with SAM4U, an SAP free customer software asset management tool.
SAM4U, an SAP complimentary software asset management tool for customers, delivers a detailed and well-structured overview of license inventory and usage with a user-friendly interface. We offer a hosted, cost-effective, and performance-optimized SAM4U setup in the Skybuffer Cloud environment. You retain ownership of the system and data, while we manage the ABAP 7.58 infrastructure, ensuring fixed Total Cost of Ownership (TCO) and exceptional services through the SAP Fiori interface.
In the rapidly evolving landscape of technologies, XML continues to play a vital role in structuring, storing, and transporting data across diverse systems. The recent advancements in artificial intelligence (AI) present new methodologies for enhancing XML development workflows, introducing efficiency, automation, and intelligent capabilities. This presentation will outline the scope and perspective of utilizing AI in XML development. The potential benefits and the possible pitfalls will be highlighted, providing a balanced view of the subject.
We will explore the capabilities of AI in understanding XML markup languages and autonomously creating structured XML content. Additionally, we will examine the capacity of AI to enrich plain text with appropriate XML markup. Practical examples and methodological guidelines will be provided to elucidate how AI can be effectively prompted to interpret and generate accurate XML markup.
Further emphasis will be placed on the role of AI in developing XSLT, or schemas such as XSD and Schematron. We will address the techniques and strategies adopted to create prompts for generating code, explaining code, or refactoring the code, and the results achieved.
The discussion will extend to how AI can be used to transform XML content. In particular, the focus will be on the use of AI XPath extension functions in XSLT, Schematron, Schematron Quick Fixes, or for XML content refactoring.
The presentation aims to deliver a comprehensive overview of AI usage in XML development, providing attendees with the necessary knowledge to make informed decisions. Whether you’re at the early stages of adopting AI or considering integrating it in advanced XML development, this presentation will cover all levels of expertise.
By highlighting the potential advantages and challenges of integrating AI with XML development tools and languages, the presentation seeks to inspire thoughtful conversation around the future of XML development. We’ll not only delve into the technical aspects of AI-powered XML development but also discuss practical implications and possible future directions.
Monitoring and Managing Anomaly Detection on OpenShift.pdfTosin Akinosho
Monitoring and Managing Anomaly Detection on OpenShift
Overview
Dive into the world of anomaly detection on edge devices with our comprehensive hands-on tutorial. This SlideShare presentation will guide you through the entire process, from data collection and model training to edge deployment and real-time monitoring. Perfect for those looking to implement robust anomaly detection systems on resource-constrained IoT/edge devices.
Key Topics Covered
1. Introduction to Anomaly Detection
- Understand the fundamentals of anomaly detection and its importance in identifying unusual behavior or failures in systems.
2. Understanding Edge (IoT)
- Learn about edge computing and IoT, and how they enable real-time data processing and decision-making at the source.
3. What is ArgoCD?
- Discover ArgoCD, a declarative, GitOps continuous delivery tool for Kubernetes, and its role in deploying applications on edge devices.
4. Deployment Using ArgoCD for Edge Devices
- Step-by-step guide on deploying anomaly detection models on edge devices using ArgoCD.
5. Introduction to Apache Kafka and S3
- Explore Apache Kafka for real-time data streaming and Amazon S3 for scalable storage solutions.
6. Viewing Kafka Messages in the Data Lake
- Learn how to view and analyze Kafka messages stored in a data lake for better insights.
7. What is Prometheus?
- Get to know Prometheus, an open-source monitoring and alerting toolkit, and its application in monitoring edge devices.
8. Monitoring Application Metrics with Prometheus
- Detailed instructions on setting up Prometheus to monitor the performance and health of your anomaly detection system.
9. What is Camel K?
- Introduction to Camel K, a lightweight integration framework built on Apache Camel, designed for Kubernetes.
10. Configuring Camel K Integrations for Data Pipelines
- Learn how to configure Camel K for seamless data pipeline integrations in your anomaly detection workflow.
11. What is a Jupyter Notebook?
- Overview of Jupyter Notebooks, an open-source web application for creating and sharing documents with live code, equations, visualizations, and narrative text.
12. Jupyter Notebooks with Code Examples
- Hands-on examples and code snippets in Jupyter Notebooks to help you implement and test anomaly detection models.
Main news related to the CCS TSI 2023 (2023/1695)Jakub Marek
An English 🇬🇧 translation of a presentation to the speech I gave about the main changes brought by CCS TSI 2023 at the biggest Czech conference on Communications and signalling systems on Railways, which was held in Clarion Hotel Olomouc from 7th to 9th November 2023 (konferenceszt.cz). Attended by around 500 participants and 200 on-line followers.
The original Czech 🇨🇿 version of the presentation can be found here: https://www.slideshare.net/slideshow/hlavni-novinky-souvisejici-s-ccs-tsi-2023-2023-1695/269688092 .
The videorecording (in Czech) from the presentation is available here: https://youtu.be/WzjJWm4IyPk?si=SImb06tuXGb30BEH .
Fueling AI with Great Data with Airbyte WebinarZilliz
This talk will focus on how to collect data from a variety of sources, leveraging this data for RAG and other GenAI use cases, and finally charting your course to productionalization.
Digital Marketing Trends in 2024 | Guide for Staying AheadWask
https://www.wask.co/ebooks/digital-marketing-trends-in-2024
Feeling lost in the digital marketing whirlwind of 2024? Technology is changing, consumer habits are evolving, and staying ahead of the curve feels like a never-ending pursuit. This e-book is your compass. Dive into actionable insights to handle the complexities of modern marketing. From hyper-personalization to the power of user-generated content, learn how to build long-term relationships with your audience and unlock the secrets to success in the ever-shifting digital landscape.
5th LF Energy Power Grid Model Meet-up SlidesDanBrown980551
5th Power Grid Model Meet-up
It is with great pleasure that we extend to you an invitation to the 5th Power Grid Model Meet-up, scheduled for 6th June 2024. This event will adopt a hybrid format, allowing participants to join us either through an online Mircosoft Teams session or in person at TU/e located at Den Dolech 2, Eindhoven, Netherlands. The meet-up will be hosted by Eindhoven University of Technology (TU/e), a research university specializing in engineering science & technology.
Power Grid Model
The global energy transition is placing new and unprecedented demands on Distribution System Operators (DSOs). Alongside upgrades to grid capacity, processes such as digitization, capacity optimization, and congestion management are becoming vital for delivering reliable services.
Power Grid Model is an open source project from Linux Foundation Energy and provides a calculation engine that is increasingly essential for DSOs. It offers a standards-based foundation enabling real-time power systems analysis, simulations of electrical power grids, and sophisticated what-if analysis. In addition, it enables in-depth studies and analysis of the electrical power grid’s behavior and performance. This comprehensive model incorporates essential factors such as power generation capacity, electrical losses, voltage levels, power flows, and system stability.
Power Grid Model is currently being applied in a wide variety of use cases, including grid planning, expansion, reliability, and congestion studies. It can also help in analyzing the impact of renewable energy integration, assessing the effects of disturbances or faults, and developing strategies for grid control and optimization.
What to expect
For the upcoming meetup we are organizing, we have an exciting lineup of activities planned:
-Insightful presentations covering two practical applications of the Power Grid Model.
-An update on the latest advancements in Power Grid -Model technology during the first and second quarters of 2024.
-An interactive brainstorming session to discuss and propose new feature requests.
-An opportunity to connect with fellow Power Grid Model enthusiasts and users.
Driving Business Innovation: Latest Generative AI Advancements & Success StorySafe Software
Are you ready to revolutionize how you handle data? Join us for a webinar where we’ll bring you up to speed with the latest advancements in Generative AI technology and discover how leveraging FME with tools from giants like Google Gemini, Amazon, and Microsoft OpenAI can supercharge your workflow efficiency.
During the hour, we’ll take you through:
Guest Speaker Segment with Hannah Barrington: Dive into the world of dynamic real estate marketing with Hannah, the Marketing Manager at Workspace Group. Hear firsthand how their team generates engaging descriptions for thousands of office units by integrating diverse data sources—from PDF floorplans to web pages—using FME transformers, like OpenAIVisionConnector and AnthropicVisionConnector. This use case will show you how GenAI can streamline content creation for marketing across the board.
Ollama Use Case: Learn how Scenario Specialist Dmitri Bagh has utilized Ollama within FME to input data, create custom models, and enhance security protocols. This segment will include demos to illustrate the full capabilities of FME in AI-driven processes.
Custom AI Models: Discover how to leverage FME to build personalized AI models using your data. Whether it’s populating a model with local data for added security or integrating public AI tools, find out how FME facilitates a versatile and secure approach to AI.
We’ll wrap up with a live Q&A session where you can engage with our experts on your specific use cases, and learn more about optimizing your data workflows with AI.
This webinar is ideal for professionals seeking to harness the power of AI within their data management systems while ensuring high levels of customization and security. Whether you're a novice or an expert, gain actionable insights and strategies to elevate your data processes. Join us to see how FME and AI can revolutionize how you work with data!
2. Factors Impacting Gene Expression Analysis
RNA Isolation
RNA integrity, purity, and yield
■■
Genomic DNA contamination
■■
Inhibitors of cDNA synthesis and qPCR
■■
RNase and DNase contamination
■■
Reagents — Reverse Transcription
cDNA synthesis efficiency
■■
RNA protection
■■
Input RNA capacity
■■
Accurate representation of mRNA
■■
Reagents — Real-Time qPCR
Detection sensitivity
■■
■■ Assay specificity
■■ Inhibitors in sample
■■ Reproducibility of thermal cycling conditions and instrument compatibility
PCR Plastic Consumables
Instrument compatibility
■■
Optimum performance
■■
Automation friendly
■■
Potential source of contamination and inhibition
■■
3. RNA Isolation
■■ Kits are designed and formulated to assist
in the isolation of highly pure and intact RNA
from different starting materials
■■ RNA is compatible with a variety of
downstream applications
– Real-time qPCR
– Northern blotting
– Microarray analysis
– cDNA library construction
■■ DNase treatment ensures genomic DNA removal
5. RNA Isolation
iScript™ RT-qPCR Sample Preparation Reagent PureZOL™ RNA Isolation Reagent
■■ Reagent stabilizes RNA and removes genomic DNA in less than 10 min ■■ Single-solution format permits recovery of RNA from small quantities of
■■ Suitable for adherent or suspension animal cells tissues or cells, making it ideally suited for gene expression studies
www.bio-rad.com/
iscript ■■ RT-qPCR is directly enabled from cells without RNA purification when
■■ Efficient RNA purification from cultured cells, yeast, viruses, and bacteria,
combined with an iScript reverse transcription kit and real-time supermix as well as plant and animal tissues
■■ Reagent allows multiplex real-time detection of up to 4 targets from as few
■■ PureZOL efficiently lyses cells and tissues, deproteinates RNA, and
as 10 cells inactivates endogenous nucleases in a single step
■■ Ideal for rapid, high-throughput gene expression analysis
■■ Scalable starting sample amount
For more information, request bulletin 5736.
■■ Convenient isolation of RNA, DNA, and protein from the same sample
104 Aurum™ Vacuum Manifold
■■ Vacuum-mediated nucleic acid purification platform
■■ Versatile manifold format adaptable for 96-well plate or up to
10 3 18 spin columns
RFU
■■ Manifold ensures fast, high-quality sample preparation while
maintaining the simplicity of vacuum processing
10 2
■■ Unique vacuum regulator design allows for complete control of
negative pressure
0 10 20 30 40
Cycles
iScript RT-qPCR sample preparation reagent generates linear
results over varying input cell amounts. HeLa cells (125, 25, 5, and
1 cells/µl) were treated and analyzed for GAPDH expression levels
using iScript cDNA synthesis kit and iQ ™ SYBR ® Green supermix
on the CFX96™ real-time PCR detection system. RFU, relative
fluorescence units.
732-6890, 100 ml
PureZOL RNA Isolation Reagent
170-8899, 5 x 10 ml 732-6470, 1 unit
iScript RT-qPCR Sample Preparation Reagent Aurum Vacuum Manifold
Amplification Reagents and Plastics 6 Visit us on the Web at www.bio-rad.com.
7. Reagents —
Reverse Transcription
■■ Formulated for efficient reverse transcription
across a broad linear dynamic range
■■ Potent RNase A inhibitors protect RNA during
setup and reverse transcription
■■ Flexible input RNA capacity to suit different
experimental needs
■■ Optimized for gene expression analysis using
real-time qPCR
9. Reagents
Two-Step Reverse Transcription Reagents
iScript™ Advanced cDNA Synthesis Kit for RT-qPCR iScript Reverse Transcription Supermix
■■Increased qPCR data throughput and cost effectiveness from a single 20 µl for RT-qPCR
www.bio-rad.com/ reverse transcription (RT) reaction ■■ 1-tube format for simple and fast setup, and reduced pipetting variability
iscript
■■ Superior sensitivity and broad linear dynamic range for RT (7.5 µg–100 fg) ■■ Liquid format at –20°C offers superior stability and eliminates freeze/thaw cycle
■■ 2-tube kit (5x iScript reaction mix and iScript reverse transcriptase) for ease ■■ Superior sensitivity and broad linear dynamic range for RT (1 µg–100 fg)
of use and reduced reaction setup time ■■ Optimized blend of oligo(dT) and random primers ensures complete and
■■ Optimized blend of oligo(dT) and random primers ensures complete and unbiased RNA sequence representation
unbiased RNA sequence representation ■■ RNase H+ MMLV reverse transcriptase (preblended with RNase inhibitor)
■■ RNase H+ MMLV reverse transcriptase (preblended with RNase inhibitor) delivers high sensitivity for real-time RT-qPCR and eliminates additional
delivers high sensitivity for real-time RT-qPCR and eliminates additional RNase H+ step
RNase H+ step ■■ Potent blend of RNaseA inhibitor protects RNA during setup and RT
■■ Potent blend of RNaseA inhibitor protects RNA during setup and RT ■■ Short 40 min protocol allows fast qPCR data generation
■■ Short 35 min protocol allows fast qPCR data generation For more information, request bulletin 6031.
For more information, request bulletin 6125.
35 104
— 1 µg RNA Average Cq SD CV, %
— 100 ng
103
104 100 ng 21.35 0.123 0.576
30 — 10 ng
RFU
— 1 ng 100 pg 31.56 0.147 0.465
102 — 100 pg
25 — 10 pg
RFU
RFU
— 1 pg
Cq
0 10 20
Cycles
30 40
103
20 103
102
15
–2 –1 0 1 2 3 4 0 10 20 30 40 10 20 30 40
log starting quantity Cycles Cycles
iScript advanced cDNA synthesis kit for RT-qPCR provides iScript reverse transcription supermix for RT-qPCR efficiently Excellent data reproducibility. PGK-1 mRNA (~160 bp), a gene that
superior sensitivity and a broad linear dynamic range for reverse reverse transcribes RNA over a broad linear dynamic range encodes a glycolytic enzyme, was quantified using iScript reverse
transcription. Total RNA (7.5 µg–1 pg) from HeLa cells was reverse for reliable gene expression analysis data. Different amounts of transcription supermix for RT-qPCR both with 100 ng () and 100 pg ()
transcribed using the iScript advanced cDNA synthesis kit for HeLa cell RNA (amounts shown in inset) were reverse transcribed of input RNA. For each input RNA, 48 individual RT reactions were
RT-qPCR in a 20 µl reaction. A tenfold dilution of generated cDNA and one-tenth of the resulting cDNA was used as a template to performed and one-tenth of the resulting cDNA was used in the qPCR
was used as template to amplify α-tubulin in a 10 µl qPCR reaction amplify β-actin gene (~90 bp) in 20 µl qPCR reactions with iQ™ reaction with SsoFast™ probes supermix. The gene expression analysis
with iQ™ SYBR® Green supermix on a CFX384™ real-time PCR SYBR® Green supermix. Standard curve R 2 = 0.999, efficiency = data show excellent reproducibility both with high and low levels of input
detection system. Efficiency = 90.7%, R2 = 0.999, slope = –3.57. Cq, 99.7%, slope = –3.33. RFU, relative fluorescence units. target mRNA. The ~10 Cq difference for the 1,000-fold dilution of RNA
quantification cycle; RFU, relative fluorescence units. (100 ng–100 pg) demonstrates good reverse transcription efficiencies
across different input RNAs. Cq, quantification cycle; RFU, relative
fluorescence units.
Amplification Reagents and Plastics 10 Visit us on the Web at www.bio-rad.com.
11. Reagents
One-Step RT-qPCR Reagents
iScript™ One-Step RT-PCR Kit with SYBR® Green Benefits of iScript one-step kits:
■■ For use on a broad range of real-time PCR instruments ■■ Provide powerful combination of iScript RNase H+ reverse transcriptase
www.bio-rad.com/
iscript
■■ Extremely sensitive detection (100 ng–1 pg) of input RNA and antibody-mediated hot-start iTaq™ DNA polymerase
Are ideal for rapid, high-throughput gene expression analysis
iScript One-Step RT-PCR Kit for Probes
■■
■■ For use with all types of hybridization probes, including dual-labeled ■■ Perform cDNA synthesis and qPCR in 1 tube, minimizing handling and
oligonucleotide probes, FRET probes, and molecular beacons contamination risk
■■ Extremely sensitive detection (1 µg–1 pg) of input RNA
For more information, request bulletin 3066.
PCR baseline-subtracted curve fit, RFU
PCR baseline-subtracted curve fit, RFU
10,000 1,000
1,000 100
100 10
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46
Cycles Cycles
iScript™ one-step RT-PCR kit with SYBR® Green provides high reproducibility and iScript one-step RT-PCR kit for probes delivers unparalleled results over an
sensitivity across a broad range of concentrations. Reactions were performed in triplicate, extremely wide dynamic range. RNA (1 µg–100 fg) isolated from HeLa cells using
along with no-template controls, using GAPDH primers and 100 ng–100 fg total HeLa RNA. the Aurum™ total RNA kit was reverse transcribed and amplified using primers to
Reactions were carried out on the iCycler iQ® real-time detection system. Standard curve β-actin and a FAM-labeled detection probe. Each dilution was performed in triplicate
r = 1.000, efficiency = 95%, slope = –3.47. RFU, relative fluorescence units. and RT-PCR was carried out on the iCycler iQ detection system. Standard curve
r = 1.000, efficiency = 97.2%, slope = –3.39. RFU, relative fluorescence units.
Ordering Information
Catalog # Description $
Two-Step Reverse Transcription Reagents One-Step RT-qPCR Reagents
170-8842 iScript Advanced cDNA Synthesis Kit for RT-qPCR, 50 x 20 μl reactions 170-8892 iScript One-Step RT-PCR Kit with SYBR Green, 50 x 50 μl reactions
170-8843 iScript Advanced cDNA Synthesis Kit for RT-qPCR, 250 x 20 μl reactions 170-8893 iScript One-Step RT-PCR Kit with SYBR Green, 200 x 50 μl reactions
170-8890 iScript cDNA Synthesis Kit, 25 x 20 μl reactions 170-8894 iScript One-Step RT-PCR Kit for Probes, 50 x 50 μl reactions
170-8891 iScript cDNA Synthesis Kit, 100 x 20 μl reactions 170-8895 iScript One-Step RT-PCR Kit for Probes, 200 x 50 μl reactions
170-8840 iScript Reverse Transcription Supermix for RT-qPCR, 25 x 20 μl reactions
170-8841 iScript Reverse Transcription Supermix for RT-qPCR, 100 x 20 μl reactions
170-8896 iScript Select cDNA Synthesis Kit, 25 x 20 μl reactions
170-8897 iScript Select cDNA Synthesis Kit, 100 x 20 μl reactions
Amplification Reagents and Plastics 12 Visit us on the Web at www.bio-rad.com.
12. Reagents —
Real-Time qPCR
■■ Patented Sso7d fusion enzyme technology
delivers higher processivity and inhibitor
tolerance
■■ Antibody-mediated hot-start polymerases
enable instant activation and higher specificity
■■ Choice of fast, standard, or universal
cycling conditions
■■ Formulated for optimal performance on a
variety of real-time instruments
Reagents
13. Reagents
Real-Time qPCR Reagents Selection Guide
SYBR® Green / EvaGreen Supermixes Probes Supermixes One-Step Kits for
RT-qPCR
SsoAdvanced™ SsoFast™ iQ™ SsoFast iTaq™ Fast iTaq™ EpiQ™ SsoFast iQ iQ SsoFast iTaq Fast iTaq iScript™ iScript
SYBR® EvaGreen® SYBR® EvaGreen SYBR® Green SYBR® Chromatin Probes Supermix Multiplex Probes Supermix Supermix One-Step One-Step
Green Supermix Green Supermix Supermix with Green SYBR® Supermix Powermix Supermix with ROX with ROX RT-PCR Kit RT-PCR Kit
Supermix Supermix with Low ROX Supermix Green with ROX with SYBR® for Probes
Real-Time PCR Instrument ROX with ROX Supermix Green
Bio-Rad
CFX96™, CFX96 Touch™,
CFX384™, CFX384 Touch™, CFX ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔
Connect™
iQ™, iQ™5, MyiQ™, MyiQ™2 ✔ ✔ ✔ ✔ ✔ ✔■ ✔ ✔ ✔
MiniOpticon™, DNA Engine
Opticon® I and II
✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔
Applied Biosystems
StepOne/StepOne Plus ◆ ◆ ◆ ◆ ◆ ✔ ✔ ◆ ◆ ◆ ✔ ◆ ✔ ◆ ◆
7500, ViiA7 ✔ ✔ ✔ ✔
7000, 7300, 7700, 7900HT ✔ ✔ ✔
Stratagene
Mx3000P, 3005P, 4000 ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔
Eppendorf
Mastercycler ep
realplex 2 or 4
✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔
QIAGEN/Corbett
Rotor-Gene 3000, 6000, Q ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔
Roche
LightCycler 480 ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔
LightCycler 1.0, 1.5, 2.0 ▲ ▲ ▲ ▲ ▲ ▲ ✔ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲
Idaho Technology
LightScanner HR-1 ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔
LightScanner 32 ▲ ▲ ▲ ▲ ▲ ▲ ✔ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲
✔ Recommended for use as is ◆ ROX reference setting must be turned “off” ▲ BSA must be added according to instrument specifications
Amplification Reagents and Plastics 14 Visit us on the Web at www.bio-rad.com.
15. Reagents
Real-Time qPCR — SYBR® Green/EvaGreen
SsoFast™ EvaGreen® Supermix SsoFast EvaGreen Supermix with Low ROX
■■ Inhibitor tolerance and enhanced processivity with Sso7d fusion polymerase ■■ Blended with ROX for optimal performance on Applied Biosystems
www.bio-rad.com/ standard and fast 7500 real-time PCR instruments
■■ Robust formulation ensures maximum efficiency, sensitivity,
supermixes
and reproducibility for qPCR For more information, request bulletin 5919.
■■ Instant polymerase activation and rapid polymerization kinetics deliver fast
qPCR results
For more information, request bulletin 5816.
104 100.000
6,000 900
800
5,000 700
10.000
600
500
RFU
4,000
400
103
RFU
RFU
∆Rn
300
3,000 1.000
200
100
2,000 0
0 10 20 30 40 0.100
Cycles
1,000
102
0 0.010
0 10 20 30 40 0 10 20 30 40 4 8 12 16 20 24 28 32 36 40
Cycles Cycles Cycles
The unique fusion polymerase in SsoFast EvaGreen supermix SsoFast EvaGreen supermix demonstrates superior inhibitor The unique fusion polymerase in SsoFast EvaGreen supermix
delivers extreme speed and generates exceptional qPCR results tolerance and direct qPCR capability from cell culture. Results with low ROX generates exceptional qPCR results on the ABI
in less than 30 min. Tenfold serial dilutions of 10 ng–100 ag cDNA show efficient amplification and detection of a spike-in control 7500 fast real-time PCR system. Tenfold serial dilutions of 10 ng–100 fg
from human spleen were used in each 20 μl reaction to detect template in the absence (■) or presence (■) of conditioned tissue cDNA from human spleen were used in each 20 μl reaction to detect
18S rRNA. 18S rRNA efficiency = 101.8%, r = 0.997. Total qPCR run culture medium using SsoFast EvaGreen supermix. Inset shows a α-tubulin. α-tubulin efficiency = 105.8%, r = 0.996. Total qPCR run
time = 29 min. RFU, relative fluorescence units. competitor’s standard qPCR reagent is able to amplify the spike-in time = 39 min (not including melt curve).
control template only in the absence (■) vs. presence (■) of culture
medium. RFU, relative fluorescence units.
Amplification Reagents and Plastics 16 Visit us on the Web at www.bio-rad.com.
17. Reagents
Real-Time qPCR — SYBR® Green/EvaGreen
Precision Melt Supermix EpiQ™ Chromatin SYBR® Green Supermix
■■ Optimized formulation containing EvaGreen dye delivers robust PCR and ■■ Robust formulation delivers superior sensitivity and efficiency for qPCR from
www.bio-rad.com/ high resolution melt (HRM) performance genomic DNA templates
supermixes
■■ Sensitive and effective discrimination of all 4 SNP classes across a broad ■■ Protocol is optimized for difficult real-time qPCR reactions for high GC amplicons
range of amplicons ■■ Supermix contains fluorescein and ROX and is compatible with all real-
■■ Accurate detection of CpG methylation status for epigenetic studies time PCR instruments except Applied Biosystems 7000, 7300, 7700,
and 7900 models (additional ROX can be added by ordering the dye
■■ Exceptional room temperature stability for high-throughput HRM studies
separately, catalog #172-5858)
■■ Reliable performance on any HRM-capable thermal cycler
Please refer to page 22 for more details about the EpiQ chromatin analysis kit.
For more information, request bulletin 6137.
For more information, request bulletin 6020.
A B
0.20 0.02 0.0
–0.1
0.15 0.00
Difference RFU
–0.2
Difference RFU
Difference RFU
0.10 –0.02
–0.3
–0.04
0.05 –0.4
–0.06 –0.5
0.00
–0.08 –0.6
–0.05
78 79 80 81 82 83 75 76 77 78 79 80 74 76 78 80 82 84
Temperature, °C Temperature, °C Temperature, °C
Precision melt supermix delivers robust HRM for single nucleotide polymorphisms (SNPs). Discrimination of class I Accurate methylation detection with precision melt supermix. Mixtures
and IV SNP genotypes are shown in panels A and B, respectively. Class I (A to G substitution) and class IV (A to T substitution) SNP of methylated and unmethylated human genomic DNA of varying ratios were
genotypes from mouse genomic DNA were analyzed using precision melt supermix. Wild type (■), heterozygote (■), and homozygous analyzed using HRM on a CFX384 real-time PCR detection system. Increasing
mutant (■) are shown in the difference plots normalized to wild-type samples. HRM analysis was performed on a CFX384™ real-time PCR amounts of methylated DNA (■, 0%; ■, 2%; ■, 5%; ■, 50%; ■, 75%; ■, 95%;
detection system and genotypes were automatically assigned by Precision Melt Analysis™ software. Amplification was carried out for 35 and ■, 100%) were analyzed for methylation of the human RARB2 gene. The
cycles. Total run time including melt curve = 150 min. RFU, relative fluorescence units. genomic region contains 7 CpG sites and is 88 base pairs in length. Total run
time including melt curve = 190 min. RFU, relative fluorescence units.
Amplification Reagents and Plastics 18 Visit us on the Web at www.bio-rad.com.
19. Reagents
Real-Time qPCR Probes
iQ™ Multiplex Powermix iTaq™ Fast Supermix with ROX
■■ Robust supermix formulated for sensitive and efficient multiplex qPCR ■■ Developed and validated for Applied Biosystems 7500 standard and fast
www.bio-rad.com/ and Stratagene real-time PCR instruments
supermixes
■■ Reliable quantification of up to 4 targets (expression levels can vary up
to 106-fold between target genes) or up to 5 targets ■■ Compatible with standard and fast thermal cycling conditions
■■ Linearity over 6 orders of magnitude of input cDNA and 4 orders of ■■ Antibody-mediated iTaq DNA polymerase provided in a convenient
magnitude of input genomic DNA 2x supermix
■■ Suitable for a wide variety of applications, including gene expression For more information, request bulletin 5737.
analysis, SNP genotyping, SNP analysis, GMO detection, and viral
load detection
iTaq Supermix with ROX
■■ Developed and validated for Applied Biosystems 7000, 7300, 7700, 7900,
For more information, request bulletin 5348.
StepOne, and StepOne Plus real-time PCR instruments
■■ Sensitive and accurate detection over 6 orders of magnitude
■■ Hot-start iTaq DNA polymerase in optimized buffer for simplex and
duplex qPCR
For more information, request bulletin 3065.
10.000
PCR baseline-subtracted curve fit, RFU
1,000
1.000
∆Rn
∆Rn
1.000
100
0.100 0.100
0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 35 40 45 0 10 20 30 40
Cycles Cycles Cycles
iQ multiplex powermix produces highly reliable qPCR results Robust duplex qPCR results with iTaq fast supermix with ROX on the iTaq supermix with ROX produces superior results on the ABI 7900HT
for up to five targets in a single tube, with no difference in Applied Biosystems 7500 fast real-time PCR system. cDNA inputs: system. Total RNA from HeLa cells was reverse transcribed using the
detection of a low-expressing gene in multiplex or singleplex. 100-fold serial dilutions of cDNA from the equivalent of 1 µg–1 pg total RNA iScript™ reverse transcription supermix for RT-qPCR. A tenfold dilution of
One-tenth of a 1 µg cDNA synthesis reaction of human thymus total were used in each 20 µl reaction. FAM-labeled 18S rRNA probe duplex generated cDNA (100 ng–1 pg) was used as template to amplify the β-actin
RNA was used in each 20 µl reaction. FAM-labeled β-actin probe (), reaction (), VIC-labeled beta-2-microglobulin (B2M) probe duplex gene with a FAM-labeled probe. Standard curve R2 = 0.999, efficiency =
Cy5-labeled α-tubulin probe (), HEX-labeled GAPDH probe (), reaction (), VIC-labeled B2M probe singleplex reaction (). 18S rRNA 98.0%, slope = –3.38.
TAMRA-labeled cyclophilin probe (), Texas Red–labeled IL-2 probe (). efficiency = 98.6%, r = 0.999; B2M efficiency = 98.0%, r = 0.998. Total
RFU, relative fluorescence units. qPCR run time = 45 min.
Amplification Reagents and Plastics 20 Visit us on the Web at www.bio-rad.com.