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.
methods of isolation and extraction of RNA by using different source such as plant tissues, bacterial culture, etc. Ribonucleic acid can be isolated from plant tissue for the purpose of:
– mRNA isolation
– In vitro translation
– Northern analysis
– cDNA library construction
Rigorous ribonuclease free environment is to be maintained
All glasswares, plasticwares and reagents made RNAse free (using 0.01% DEPC)
Next day, DEPC is inactivated by autoclaving for 30 min
Total RNA is isolated and separated from DNA and protein after extraction with a solution called as Trizol. Trizol is an acidic solution containing guanidinium thiocyanate (GITC), phenol and chloroform. GITC irreversibly denatures proteins and RNases. This is followed by centrifugation.
There are 'n' number of DNA isolation methods depending on the sample type, final use of DNA product, etc. This presentation gives an overall idea about different methods of DNA isolation in a simplified way.
methods of isolation and extraction of RNA by using different source such as plant tissues, bacterial culture, etc. Ribonucleic acid can be isolated from plant tissue for the purpose of:
– mRNA isolation
– In vitro translation
– Northern analysis
– cDNA library construction
Rigorous ribonuclease free environment is to be maintained
All glasswares, plasticwares and reagents made RNAse free (using 0.01% DEPC)
Next day, DEPC is inactivated by autoclaving for 30 min
Total RNA is isolated and separated from DNA and protein after extraction with a solution called as Trizol. Trizol is an acidic solution containing guanidinium thiocyanate (GITC), phenol and chloroform. GITC irreversibly denatures proteins and RNases. This is followed by centrifugation.
There are 'n' number of DNA isolation methods depending on the sample type, final use of DNA product, etc. This presentation gives an overall idea about different methods of DNA isolation in a simplified way.
Effective disruption of the biological matrix (cell, tissue, environmental or biological sample) to release the nucleic acids. Denaturation of structural proteins associated with the nucleic acids (nucleoproteins) Inactivation of nucleases that will degrade the isolated product (RNase and/or DNase).
Once the genomic DNA is bound to the silica membrane, the nucleic acid is washed with a salt/ethanol solution. These washes remove contaminating proteins, lipopolysaccharides and small RNAs to increase purity while keeping the DNA bound to the silica membrane column.
There are five basic steps of DNA extraction that are consistent across all the possible DNA purification chemistries:
disruption of the cellular structure to create a lysate,
separation of the soluble DNA from cell debris and other insoluble material,
binding the DNA of interest to a purification matrix,
washing proteins and other contaminants away from the matrix and
elution of the DNA.
Techniques of DNA Extraction, Purification and QuantificationBHUMI GAMETI
Introduction
The overall process…
Uses of isolated genomic DNA
Extraction of DNA from plant material
Components of DNA extraction solutions
Cell Lysis or Cell disruption :
Purification of DNA
CTAB Method
Phenol–chloroform extraction
PROTEINASE K
Salting out
Silica adsorption method
Magnetic beads
FTA Paper
Nucleic acid quantification
Agarose Gel Electrophoresis
UV spectroscopy
DNA quantification using NanoDrop
Esta presentación la encontré en la web y entiendo que es muy útil para la práctica de Electroforesis de Gel Agarosa. Explica de forma básica de que consta esta tecnica molecular y los pasos a seguir.
b pharma 6th sem
nucleic acid extraction and quantification
pharmaceutical biotechnology
Introduction
Purpose
Isolation
Methods of isolation
Basic steps for DNA extraction
Organic extraction
Inorganic extraction
salting out
DNA Fingerprinting of plants . History,procedure of DNA fingerprinting, PCR and NON PCR technique like RAPD,SSR,RELPs, application of DNA fingerprinting, advantage and disadvantage of DNA fingerprinting.
Effective disruption of the biological matrix (cell, tissue, environmental or biological sample) to release the nucleic acids. Denaturation of structural proteins associated with the nucleic acids (nucleoproteins) Inactivation of nucleases that will degrade the isolated product (RNase and/or DNase).
Once the genomic DNA is bound to the silica membrane, the nucleic acid is washed with a salt/ethanol solution. These washes remove contaminating proteins, lipopolysaccharides and small RNAs to increase purity while keeping the DNA bound to the silica membrane column.
There are five basic steps of DNA extraction that are consistent across all the possible DNA purification chemistries:
disruption of the cellular structure to create a lysate,
separation of the soluble DNA from cell debris and other insoluble material,
binding the DNA of interest to a purification matrix,
washing proteins and other contaminants away from the matrix and
elution of the DNA.
Techniques of DNA Extraction, Purification and QuantificationBHUMI GAMETI
Introduction
The overall process…
Uses of isolated genomic DNA
Extraction of DNA from plant material
Components of DNA extraction solutions
Cell Lysis or Cell disruption :
Purification of DNA
CTAB Method
Phenol–chloroform extraction
PROTEINASE K
Salting out
Silica adsorption method
Magnetic beads
FTA Paper
Nucleic acid quantification
Agarose Gel Electrophoresis
UV spectroscopy
DNA quantification using NanoDrop
Esta presentación la encontré en la web y entiendo que es muy útil para la práctica de Electroforesis de Gel Agarosa. Explica de forma básica de que consta esta tecnica molecular y los pasos a seguir.
b pharma 6th sem
nucleic acid extraction and quantification
pharmaceutical biotechnology
Introduction
Purpose
Isolation
Methods of isolation
Basic steps for DNA extraction
Organic extraction
Inorganic extraction
salting out
DNA Fingerprinting of plants . History,procedure of DNA fingerprinting, PCR and NON PCR technique like RAPD,SSR,RELPs, application of DNA fingerprinting, advantage and disadvantage of DNA fingerprinting.
RNA Integrity and Quality – Standardize RNA Quality Control QIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the challenges and considerations of handling RNA samples, the need for quality control analysis and common methods for RNA integrity and quality assessment. The QIAxcel Advanced System will be introduced to automate the process of RNA sample integrity analysis and obtain objective quality measurement. Application data will be presented.
Genomic library and shotgun sequencing. It includes the topics about genomic library,construction method, its uses and applications, shotgun sequencing, difference between random and whole genome sequencing, its advantages and disadvantages etc.
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.
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.
How to Increase the Quality and Quantity of your RNA Extractions.pdffateh11
Posted: January 14, 2013
How to Increase the Quality and Quantity of your RNA Extractions
When isolating RNA, good lab technique is essential. RNA is more labile in comparison to fixed and
sacrosanct DNA. RNA, with a ribose sugar backbone, contains highly reactive hydroxyl (C-OH) groups,
ensuring that strands are continually made, broken down and reused. Isolated RNA quickly degrades and
is easily contaminated by resistant enzymatic ribonucleases (RNases), which are found everywhere.
In this edition of Bench Tips, we spoke with Dr. Jack Dumbacher, chairman and curator in the
Department of Ornithology and Mammalogy at the California Academy of Sciences in San Francisco,
about his lab’s current work isolating transcriptome and small RNA for the identification of metagenomic
viral sequences. Contrary to the typical view of viruses as pathogens, viruses may also have mutualistic or
symbiotic relationships with their hosts. Understanding these evolutionary relationships in birds is one of
Dumbacher’s research goals.
“Typically with virus particles you’re after low-abundance RNA,” says Dumbacher. “Ribosomal RNA
(rRNA) can be a huge portion of what you initially pick up. For good recovery, we try to increase our
RNA yield by taking big samples in the field and preserving them as carefully as possible.”
Optimize RNA preservation
The Academy’s collecting expeditions often take place in remote areas renowned for their biodiversity,
such as tropical forests or coral reefs. A big challenge for Dumbacher’s team is the inability to snap-freeze
samples with liquid nitrogen because of the lack of refrigeration. (RNA steadily degrades in ethanol, a
common preservative for DNA samples.) Instead samples—swabbed from the bird’s cloaca, the rear
digestive opening—are placed in RNA stabilization reagents or other mixtures containing guanidinium
thiocyanate (GITC) buffer at a 4 M concentration. RNA isolation in liquid samples can be difficult using
some RNA stabilization reagents because of the salt content. When maintaining a chain of refrigeration is
not an issue, the best way to preserve RNA is to immediately freeze samples after collection with liquid
nitrogen and store samples at -80°C.
Ensure good separation
Traditionally, GITC buffers have been used to lyse cells and viral particles in RNA extractions and
simultaneously prevent RNase enzymatic activity by denaturing RNA temporarily. In such cases, some
intact viral separation—like filtration—must be performed before lysing to avoid a soup of nucleic acids.
The samples collected by the Dumbacher team are predominantly in liquid form—little homogenization is
required, but good separation is important. In the field, the team uses 0.2 µ filters—similar to those found
in spin columns—to filter through smaller virus particles. Larger particles, like intact cells, are left
behind. Other labs work with frozen tissue samples that must be homogenized quickly and thoroughly for
good recovery. Regardless of the chosen metho
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.
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.
Optimal RNAlater® incubation and removal conditions prior to isolation of tot...QIAGEN
RNA is highly sensitive to degradation. Handling methods and prolonged storage of cells can greatly affect the quality of the RNA that can be later isolated. Contamination with RNases is the most significant problem, especially as they are so ubiquitous in the environment. They can degrade RNA to the point where results of downstream analyses become meaningless.
Submerging cells in RNAlater, an RNA stabilization reagent, helps to stabilize the RNA within the cells and prevent degradation, supporting accurate downstream gene expression analyses. However, to avoid any interference from any RNAlater components in isolation and analyses, cells must be pelleted and the reagent must be removed. The separation of cells from excess RNAlater via centrifugation is impeded due to the higher density of the reagent compared to standard culture medium. This means it requires higher centrifugal forces, which might damage cells due to increased shearing forces, leading to reduced RNA yield. The aim of this study was to establish the optimal conditions for the recovery of cells from RNAlater after RNA stabilization for maximum RNA yield and integrity.
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.
In this slidedeck, the following topics, which are critical steps for efficient and precise gene expression studies using real-time PCR technology, are covered:
• Effect of RNA integrity on real-time PCR results – tips on how to achieve a true RNA profile suitable for real-time PCR studies
• Improved methods for cDNA synthesis, optimized for real-time PCR
• Real-time PCR analysis
• Real-time PCR essentials and background information on different quantification strategies
• SYBR Green real-time PCR – factors influencing specificity
• Introduction to probe technology
• New, fast and efficient real-time PCR solutions
RNA analysis on non-denaturing agarose gel electrophoresis.pdffateh11
use 1X TAE buffer instead of 1X TBE
- use agarose gel in the concentration of 1.1%-1.2%
- add ethidium bromide (EtBr) to the gel and electrophoresis buffer to avoid the additional (potentially
RNAse-prone) step of gel staining
- always use fresh gel and buffer as well as clean electrophoresis equipment for RNA analysis. Wear
gloves to protect RNA samples from degradation by nucleases and avoid a hand contact with EtBr. - use
running voltage up to 10 V/cm (10V per each cm of space between the electrodes in electrophoretic
chamber). Do not use high voltage to avoid RNA degradation during electrophoresis.
2. Heat an aliquot of the RNA solution at 70°C for 1 min and place it on ice before loading on a gel.
3. Load a known amount of DNA or RNA ladder alongside your RNA sample as a standard for
determining the RNA concentration. RNA concentration can be roughly estimated assuming that the
efficiency of EtBr incorporation in rRNA is the same as for DNA (the ribosomal RNA may be considered
a double-stranded molecule due to its extensive secondary structure).
4. The first sign of RNA degradation on the non-denaturing gel is a slight smear starting from the rRNA
bands and extending to the area of shorter fragments. RNA showing this extent of degradation is still
good for further procedures. However, if the downward smearing is so pronounced that the rRNA bands
do not have a discernible lower edge, this RNA should be discarded.
The following characteristics indicate successful RNA preparation:
- For mammalian total RNA, two intensive bands should be observed against a light smear. These bands
represent 28S and 18S rRNA. The ratio of intensities of these bands should be about 1.5-2.5:1. Intact
mammalian poly (A)+ RNA appears as a smear sized from 0.1 to 4-7 (or more) kb with faint 28S and 18S
rRNA bands.
- In the case of RNA from non-mammalian sources (plants, insects, yeast, amphibians), the normal
mRNA smear on the non-denaturing agarose gel may not exceed 2-3 kb. Moreover, the overwhelming
majority of invertebrates have 28s rRNA with a so-called "hidden break" (Ishikawa, 1977). In some
organisms the interaction between the parts of 28s rRNA is rather weak, so the total RNA preparation
exhibits a single 18s-like rRNA band even on a non-denaturing gel. In other species the 28s rRNA is more
robust, so it is still visible as a second band.
Note: If your experimental RNA is shorter than expected and/or degraded according to electrophoresis
data, prepare fresh RNA after checking the quality of RNA purification reagents. If problems persist, you
may need to find another source of tissue/cells. In some cases, partially degraded RNA is only available
(e.g. tumor samples or hard treated tissues). This RNA can be used for cDNA preparation, however the
cDNA sample will contain reduced number of full-length molecules.
Total RNA from endothelial human cells.
- Commonly, genomic DNA contamination does not exceed the amount seen on the agarose/EtBr gel as a
weak band of
Genomics research and discovery has led to a large increase of reported single nucleotide polymorphisms (SNPs). From 2006 to 2017, the number of refSNPs in the NCBI dbSNP database has increased 13-fold. Many polymorphisms can be linked to disease susceptibility and responses to chemical therapies. Other polymorphisms are used as trait identifiers in livestock and plants. Being able to inexpensively and accurately determine the genotype in high-throughput fashion, with low sample input is a critical need in current, large-scale screening efforts. In this presentation, we present a novel, probe-based, PCR genotyping solution that possesses the universal cycling conditions, strong signal generation, and benchtop reaction stability needed for high-throughput screening. We also present the mechanism and unique technical advantages of using the rhAmp SNP Genotyping System, and we will illustrate how easy it is to generate high quality genotyping data.
Hot-start DNA polymerases are commonly used in PCR for genotyping, sequencing, molecular diagnostics, and high-throughput applications. In this presentation, PCR performance of Invitrogen™ Platinum II Taq Hot-Start DNA Polymerase and Invitrogen™ AccuPrime Taq DNA Polymerase is compared in the following areas:
• PCR run time for targets of different lengths
• Amplification of AT-rich and GC-rich sequences
• Tolerance to PCR inhibitors
• Sensitivity in target detection
• Universal protocol for PCR targets of different lengths
• Multiplex PCR of 15 targets
• Product format for direct gel loading
Request a sample of Platinum II Taq enzyme at http://bit.ly/2M4U9cw
Find other PCR enzymes at http://bit.ly/2JIPrzj
Learn more about PCR at http://bit.ly/2y2aSVo
#PCR #PCREducation #Invitrogen #InvitrogenSchoolofMolBio
Human cytomegalovirus (CMV) is a common immune-evasive herpes family virus leading to lifelong asymptomatic infection in 50 to 80% of humans. Current research evaluating the use of
TCR sequencing to predict response to immunotherapy has focused on measurements of T cell clonal expansion and TCR convergence (2,3,4) as potential predictive biomarkers for
response. Given that CMV infection has been reported to elicit large clonal proliferations of CMV reactive T cells (1), and is a source of chronic antigen stimulation, we hypothesized that CMV
infection might alter T cell repertoire features in a manner relevant to the potential biomarker use of TCR sequencing. Here we sought to identify features of CMV infection using TCRB profiling of
peripheral blood (PBL) total RNA. We identify reduced T cell evenness and elevated TCR convergence as features of chronic CMV infection.
Improvement of TMB Measurement by removal of Deaminated Bases in FFPE DNAThermo Fisher Scientific
Tumor mutational burden (TMB) is a positive predictive factor for response to immune-checkpoint inhibitors in certain types of cancer. The Oncomine™ Tumor Mutation Load Assay, a targeted next generation sequencing (NGS) assay, measures TMB (from 1.2Mb of coding region) and detects mutations in 409 cancer genes. The TMB values obtained using targeted sequencing are highly correlated with TMB measured by whole exome sequencing. FFPE preservation methods can lead to significant cytosine deamination of the isolated DNA, resulting in decreased sequencing quality. In these samples, uracils are propagated as thymines and result in false C>T substitutions. Analysis of the Oncomine™ TML Assay using Torrent Suite and Ion Reporter ™ software uniquely estimates the degree of deamination in fixed tissues by measuring C:G>T:A variants. This deamination score is used to assess quality of DNA extracted from FFPE tumor tissue. To minimize the influence
that excess deamination has on TMB results, we have incorporated a repair treatment to eliminate damaged targets and improve usable TMB values of DNA from damaged FFPE tumor tissue using Uracil-DNA glycosylase (UDG). The
Oncomine™ TML Assay for TMB on the Ion Gene Studio™ S5 systems in conjunction with a deamination score is informative and potentially predictive for the use of checkpoint inhibitors in multiple cancer types.
What can we learn from oncologists? A survey of molecular testing patternsThermo Fisher Scientific
Oncologists are increasingly incorporating NGS testing to guide targeted and immuno-oncology therapies1. Most clinical NGS testing is confined to large academic institutions and reference labs, despite the fact that most cancer patients are treated in the community settings. We therefore sought to examine molecular testing selection patterns directly from oncologists in order to better identify perceived gaps in testing and treatment paradigms
Evaluation of ctDNA extraction methods and amplifiable copy number yield usin...Thermo Fisher Scientific
The use of cell-free circulating tumor DNA (ctDNA) for non-invasive cancer testing has the potential to revolutionize the field. However, emergence of an increasing number of extraction methods and detection assays is rendering laboratory workflow development much more complex and cumbersome. The use of standardized, well characterized ctDNA control materials in human plasma could facilitate the evaluation of extraction efficiency and assay performance across platforms. In this study, we use a full process ctDNA quality control material in true human plasma to demonstrate the variability of extraction yield between different ctDNA extraction kits. We also examine the correlation between the amplifiable
copy number and DNA concentration post-extraction.
Analytical Validation of the Oncomine™ Comprehensive Assay v3 with FFPE and C...Thermo Fisher Scientific
Presented here is an analytical validation of OCAv3 at the Life Technologies Clinical Services Laboratory (LTCSL), a CAP-accredited and CLIA-certified clinical laboratory. Analytical validations provide evidence of consistently accurate and relevant sequencing results.
Novel Spatial Multiplex Screening of Uropathogens Associated with Urinary Tra...Thermo Fisher Scientific
Accurate identification of uropathogens in a timely manner is important to correctly understand urinary tract infections(UTI’s), which affects nearly 150 million people each year. The
current standard approach for detecting the UTI pathogens is culture based. This method is time consuming, has low throughput, and can lack sensitivity and/or specificity. In addition, not all uropathogens grow equally well under standard culture conditions which can result in a failure to detect the species. To address these gaps, we have developed a unique workflow from sample preparation to target identification using the nanofluidic OpenArray™ platform for spatial multiplexing of target specific assays. In this study, we tested pre-determined blinded research samples and confirmed the subset of results with orthogonal Sanger sequences.
Liquid biopsy quality control – the importance of plasma quality, sample prep...Thermo Fisher Scientific
Liquid biopsy is emerging as a non-invasive companion to traditional solid tumor biopsies. As next generation sequencing (NGS) of circulating cell-free nucleic acids (cfNA = cfDNA and cfRNA) becomes common, it’s important to understand the impact of sample preparation on quality, specificity, and sensitivity of liquid biopsy tests. Plasma samples are often limited, and may have undesirable characteristics such as lipemia or hemolysis that contribute unwanted genomic DNA (gDNA) to the sample. Low cfDNA concentration can also limit the amount available for NGS library prep. In this study, we explore the effects of suboptimal plasma and low library input on liquid biopsy NGS, and discuss various techniques for in-process quality control of cfNA samples isolated from plasma
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
Targeted T-cell receptor beta immune repertoire sequencing in several FFPE ti...Thermo Fisher Scientific
T-cell receptor beta (TCRβ) immune repertoire analysis by next-generation sequencing is a valuable tool for studies of the tumor microenvironment and potential immune responses to cancer immunotherapy. Here we describe a TCRβ sequencing assay that leverages the low sample input requirements of AmpliSeq library preparation technology to extend the capability of targeted immune repertoire sequencing to include FFPE samples which can often be degraded and in short supply
Development of Quality Control Materials for Characterization of Comprehensiv...Thermo Fisher Scientific
Targeted next-generation sequencing (NGS) panels can detect hundreds of mutations in key genes using amplification based and hybrid-capture based NGS technologies. Although NGS technology is a powerful tool, optimizing and characterizing test performance on hundreds of variants is extremely challenging, time consuming, and expensive. Samples must be sourced, variants identified and orthogonally confirmed, then quantified and diluted. This effort is then multiplied across dozens of samples, and then samples must be run over many runs and days to assess assay reproducibility, precision, sensitivity, etc. In this study, we developed a novel reference material, experimental design, and analysis pipeline that allows for highly streamlined NGS assay characterization, enabling thorough test characterization across 500+ variants within only 6 runs.
As one of the leading causes of death globally, respiratory
infections could be caused by single or multiple types of viral,
bacterial or fungal pathogens that present in the upper and
lower respiratory tract. Panel-based testing using molecular
methods to identify multiple pathogens simultaneously can
contribute to better understanding of respiratory infections.
A high-throughput approach for multi-omic testing for prostate cancer researchThermo Fisher Scientific
The proliferation of genetic testing technologies and genome-scale studies has increased our understanding of the genetic basis of complex diseases. However, this information alone tells an incomplete story of the underlying biology. Integrative approaches that combine data from multiple sources, such as the genome, transcriptome and/or proteome, can provide a more comprehensive and multi-dimensional model of complex diseases. Similarly, the integration of multiple data types in disease screening can improve our understanding of disease in populations. In a series of groundbreaking multi-omic, population-based studies of prostate cancer, researchers at the Karolinska Institutet in Stockholm, Sweden identified sets of genetic and protein biomarkers that when evaluated together with other clinical research data performed significantly better in predicting cancer risk (1,2) than the most-widely used single protein biomarker, the prostate-specific antigen (PSA).
Discover the innovations and more that led to amazing discoveries through the use of thermal cyclers. What were scientists able to accomplish? What things are important to them when selecting a thermal cycler? What do you need to advance your science?
Learn more about thermal cyclers: http://bit.ly/2Q2oPhF
See all thermal cycler offerings: http://bit.ly/2Paf1wH
A rapid library preparation method with custom assay designs for detection of...Thermo Fisher Scientific
Herein, we describe a new research method for library
preparation using the Ion AmpliSeq™ HD Library Kit with
custom assay designs from Ion AmpliSeq HD Panels for
detection of low level variants from liquid biopsy samples. This
method includes incorporation of molecular tags that enable
0.1% Limit of Detection (LOD) in cell free DNA (cfDNA) and
dual barcodes for sample identification. This method is also
applicable to formalin-fixed paraffin embedded (FFPE)
samples. The libraries can be prepared in as little as 3 hours
and are compatible for analysis with the Ion GeneStudio™ S5
system
Generation of Clonal CRISPR/Cas9-edited Human iPSC Derived Cellular Models an...Thermo Fisher Scientific
Reprogramming permits the derivation of hiPSCs from diseased patients, and allows us to model diseases in vitro. Furthermore, with the advent of CRISPR mediated genome editing, we can now mimic disease mutations in control hiPSC lines to study the biological effect of just those mutations. hiPSCs can then be differentiated into specified cell types such as neurons which can be used to develop assays for drug safety screening or can be used to model disease phenotypes in a dish to discover new drugs.
TaqMan®Advanced miRNA cDNA synthesis kit to simultaneously study expression o...Thermo Fisher Scientific
MicroRNAs (miRNA) are a class of small non-coding RNAs (approximately 21 nt long) that bind complementary sequences in target mRNAs to specifically regulate gene expression. Aberrant regulation of miRNAs and their targets has been associated with several diseases including cancer. The relationship between miRNA and mRNA has been found to be important in cancer development and progression. Simultaneous expression studies of miRNA and mRNA and detection of mutations in mRNA transcripts can be valuable in understanding molecular mechanisms that
have an underlying role in various diseases. We demonstrate the technical verification of a novel method to reverse-transcribe and pre-amplify miRNA and mRNA from sample-limiting serum research samples using the TaqMan® Advanced miRNA cDNA Synthesis Kit. Based on results from previous studies, a signature of 49 mRNA and 37 miRNA targets has been identified that may help distinguish between benign and malignant pancreatic tissues. In this study, these targets and an additional set of transcript mutations were analyzed in serum from normal and test samples. TaqMan assays for miRNA and mRNA targets and custom TaqMan Mutation Detection Assays (TMDAs) were placed on TaqMan Array Cards to facilitate investigation of several samples in a single experiment. Results demonstrate that transcript mutations can be detected and miRNA and mRNA targets can be reliably quantified from a single reverse transcription reaction. For research use only. Not for use in diagnostic purposes.
Identifying novel and druggable targets in a triple negative breast cancer ce...Thermo Fisher Scientific
In this study, we developed a CRISPR/Cas9-based high throughput loss-of-function screen for identifying target genes responsible for the tumor proliferation and growth in TNBC. Our initial focus was to identify essential kinases in MDA-MB-231 cell line using the Invitrogen™ LentiArray™ Human Kinase CRISPR Library, which targets 840 kinases with up to 4 different gRNAs per protein kinase for complete gene knockout. This functional screen identified over 90 protein kinases that are essential for cell viability and cell proliferation. Ten of these hits (CDK1, CDK2, CDK8, CDK10, CDK11A, CDK19, CDK19, CDC7, EPHA2 and WEE1) are well-known targets validated in the literature. Currently, we are in the process validating the novel hits through target gene sequencing, western blotting and target specific small molecule kinase inhibitors.
Evidence for antigen-driven TCRβ chain convergence in the melanoma-infiltrati...Thermo Fisher Scientific
T cell convergence refers to the phenomenon whereby antigen-driven selection enriches for T cell receptors (TCRs) having a shared antigen specificity but different amino acid or
nucleotide sequence. T cell recruitment and expansion within the tumor microenvironment (TME) may be directed by responses to tumor neoantigen, suggesting that elevated T
cell convergence could be a general feature of the tumor infiltrating T cell repertoire. Here we use the Ion AmpliSeq™ Immune Repertoire Assay Plus – TCRβ to evaluate evidence
for T cell convergence within melanoma tumor biopsy research samples from a set of 63 subjects plus peripheral blood leukocytes (PBL) from four healthy subjects. We find that the melanoma TME is highly enriched for convergent TCRs compared to healthy donor peripheral blood. We discuss the potential use of TCR convergence as a liquid biopsy compatible predictive biomarker for immunotherapy response.
Analytical performance of a novel next generation sequencing assay for Myeloi...Thermo Fisher Scientific
To support clinical and translational research into precision oncology strategies for myeloid cancers, a next-generation sequencing (NGS) assay was developed to detect common and relevant somatic alterations. To define gene targets that were recurrently altered in myeloid cancers and relevant for clinical and translational research, an extensive survey of investigators at hematology oncology research labs was performed.
The Roman Empire A Historical Colossus.pdfkaushalkr1407
The Roman Empire, a vast and enduring power, stands as one of history's most remarkable civilizations, leaving an indelible imprint on the world. It emerged from the Roman Republic, transitioning into an imperial powerhouse under the leadership of Augustus Caesar in 27 BCE. This transformation marked the beginning of an era defined by unprecedented territorial expansion, architectural marvels, and profound cultural influence.
The empire's roots lie in the city of Rome, founded, according to legend, by Romulus in 753 BCE. Over centuries, Rome evolved from a small settlement to a formidable republic, characterized by a complex political system with elected officials and checks on power. However, internal strife, class conflicts, and military ambitions paved the way for the end of the Republic. Julius Caesar’s dictatorship and subsequent assassination in 44 BCE created a power vacuum, leading to a civil war. Octavian, later Augustus, emerged victorious, heralding the Roman Empire’s birth.
Under Augustus, the empire experienced the Pax Romana, a 200-year period of relative peace and stability. Augustus reformed the military, established efficient administrative systems, and initiated grand construction projects. The empire's borders expanded, encompassing territories from Britain to Egypt and from Spain to the Euphrates. Roman legions, renowned for their discipline and engineering prowess, secured and maintained these vast territories, building roads, fortifications, and cities that facilitated control and integration.
The Roman Empire’s society was hierarchical, with a rigid class system. At the top were the patricians, wealthy elites who held significant political power. Below them were the plebeians, free citizens with limited political influence, and the vast numbers of slaves who formed the backbone of the economy. The family unit was central, governed by the paterfamilias, the male head who held absolute authority.
Culturally, the Romans were eclectic, absorbing and adapting elements from the civilizations they encountered, particularly the Greeks. Roman art, literature, and philosophy reflected this synthesis, creating a rich cultural tapestry. Latin, the Roman language, became the lingua franca of the Western world, influencing numerous modern languages.
Roman architecture and engineering achievements were monumental. They perfected the arch, vault, and dome, constructing enduring structures like the Colosseum, Pantheon, and aqueducts. These engineering marvels not only showcased Roman ingenuity but also served practical purposes, from public entertainment to water supply.
The French Revolution, which began in 1789, was a period of radical social and political upheaval in France. It marked the decline of absolute monarchies, the rise of secular and democratic republics, and the eventual rise of Napoleon Bonaparte. This revolutionary period is crucial in understanding the transition from feudalism to modernity in Europe.
For more information, visit-www.vavaclasses.com
Acetabularia Information For Class 9 .docxvaibhavrinwa19
Acetabularia acetabulum is a single-celled green alga that in its vegetative state is morphologically differentiated into a basal rhizoid and an axially elongated stalk, which bears whorls of branching hairs. The single diploid nucleus resides in the rhizoid.
Macroeconomics- Movie Location
This will be used as part of your Personal Professional Portfolio once graded.
Objective:
Prepare a presentation or a paper using research, basic comparative analysis, data organization and application of economic information. You will make an informed assessment of an economic climate outside of the United States to accomplish an entertainment industry objective.
Model Attribute Check Company Auto PropertyCeline George
In Odoo, the multi-company feature allows you to manage multiple companies within a single Odoo database instance. Each company can have its own configurations while still sharing common resources such as products, customers, and suppliers.
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
Honest Reviews of Tim Han LMA Course Program.pptxtimhan337
Personal development courses are widely available today, with each one promising life-changing outcomes. Tim Han’s Life Mastery Achievers (LMA) Course has drawn a lot of interest. In addition to offering my frank assessment of Success Insider’s LMA Course, this piece examines the course’s effects via a variety of Tim Han LMA course reviews and Success Insider comments.
Francesca Gottschalk - How can education support child empowerment.pptxEduSkills OECD
Francesca Gottschalk from the OECD’s Centre for Educational Research and Innovation presents at the Ask an Expert Webinar: How can education support child empowerment?
Read| The latest issue of The Challenger is here! We are thrilled to announce that our school paper has qualified for the NATIONAL SCHOOLS PRESS CONFERENCE (NSPC) 2024. Thank you for your unwavering support and trust. Dive into the stories that made us stand out!
Biological screening of herbal drugs: Introduction and Need for
Phyto-Pharmacological Screening, New Strategies for evaluating
Natural Products, In vitro evaluation techniques for Antioxidants, Antimicrobial and Anticancer drugs. In vivo evaluation techniques
for Anti-inflammatory, Antiulcer, Anticancer, Wound healing, Antidiabetic, Hepatoprotective, Cardio protective, Diuretics and
Antifertility, Toxicity studies as per OECD guidelines
1. Basic tips & tricks for
working with RNA
Based on Frequently Asked Questions raised by scientists
2. tip #1 Inactivate endogenous RNases
tip #2 Proper sample storage conditions
tip #3 Thorough sample homogenization
tip #4 Optimal RNA isolation
tip #5 Eliminate contaminating gDNA
tip #6 Exposure to environmental RNases
tip #7 Assess RNA quality
tip #8 Storage of RNA
tip #9 Integrate into workflow
tip #10 Experimental design
4. tip #1
Inactivate endogenous RNases
- Should be done immediately upon sample harvesting:
Homogenize samples in a chaotrope-based cell lysis solution such
as TRIzol®. This will effectively inhibit RNase activity while disrupting cells
and cell components during sample homogenization.
Flash-freeze samples in liquid nitrogen / dry-ice alcohol bath.
Tissue pieces must be small enough to freeze immediately upon immersion.
Place samples in RNAlater® Tissue Collection & RNA Stabilization
Solution. This aqueous, nontoxic reagent stabilizes and protects cellular
RNA in intact, unfrozen tissue and cell samples. Tissue samples must be in
thin pieces (≤0.5 cm thick) to allow the solution to quickly permeate before
RNases destroy the RNA.
6. tip #2
Proper sample storage conditions
-Flash-frozen sample must be stored at –80°C, even brief thawing prior to
homogenization/lysis can result in RNA degradation and loss.
-Flash-frozen tissue should be ground/pulverized at cryogenic
temperatures prior to homogenization in a lysis solution .
-RNAlater® Solution offers great flexibility for storage. Cells or tissues can
be harvested and stored at:
room temperature for up to 1 week
4°C for up to 1 month
long term at –20°C
8. tip #3
Thorough sample homogenization
-Homogenization methods should be tailored to the specific cell/tissue type:
Most cultured cells can be homogenized by vortexing in lysis
solution.
Animal/plant tissues, yeast, and bacteria require more rigorous
disruption, such as a rotor-stator homogenizer or a French Press.
Lysis of single-celled organisms is often aided by the addition of
hydrolytic enzymes specific for cell wall components to achieve maximum
recovery of RNA.
10. tip #4
Optimal RNA isolation
-Depends on sample type, application, throughput and personal preferences:
Solid-phase extraction to a thick filter matrix (column), such as the
PureLink™ RNA Mini Kit.
For tissue rich in e.g. nucleases (pancreas) or fat (brain/adipose tissue),
or applications demanding protein removal, use the TRIzol® Plus RNA
Purification System. Chaotropic salts and a strong denaturing solvent
strips off proteins. Can be used with a second purification.
Magnetic separation technology for high throughput requirements,
manual or automated methods:
• MagMAX™ nucleic acid isolation kits for genomic and viral nucleic acid from diverse
samples (cells, tissue, blood and cell-free media).
• Dynabeads® Oligo(dT)25 is available alone, and as part of different kits for isolation of mRNA
directly from virtually any crude lysate.
12. tip #5
Eliminate contaminating gDNA
-RNA for use in PCR, real-time PCR or array hybridizations should be treated
with DNase, as the DNA can interfere with the RNA signal.
-RNA from DNA-rich tissue (e.g. spleen) should also be treated with DNase .
-PureLink™ DNase Set (lyophilized RNase-free DNase) removes DNA from
RNA purified using PureLink™ RNA kits. Optimized for on-column digestion of
DNA using PureLink™ protocols.
-Ambion®’s Turbo DNase-free kit™ removes DNA in just 5 minutes.
14. tip #6
Exposure to environmental RNases
-Ensure no RNases are introduced into RNA preparations once they are no
longer protected by the strong protein denaturants used for extraction.
-RNases are found almost everywhere!
-Any item that may come into contact with the RNA must be RNase-free:
Decontaminate all surfaces (e.g. pipettes, bench-tops, glassware, and
gel equipment) with a surface decontamination solution such as
RNaseZap® or RNaseZap® Wipes.
Change gloves frequently.
Always use RNase-free tips, tubes, and solutions.
16. tip #7
Assess RNA quality
-UV absorbance readings will measure anything that absorbs at 260 nm (incl.
DNA, protein, degraded nucleic acids, and free nucleotides).
-Qubit® 2.0 Fluorometer uses fluorescent dyes to quantitate, and can
distinguish between RNA and the different contaminants . This generates
more accurate and precise results across a lower concentration range.
-Electrophoretic analysis of total RNA to assess the rRNAs. The relative area
of the large subunit (23-28S, depending on species) to the small subunit (16-
18S) is a good indication, as the larger rRNA will degrade faster.
-E-Gel® EX gels for RNA sample integrity analysis.
-For total RNA samples analyzed on an Agilent 2100 Bioanalyzer, the
software can be used to determine the RIN value (RNA Integrity Number) on
a scale of 1-10, where 10 is ideal .
-For fractionated RNA samples (e.g. poly(A)+ RNA), the type of sample must
be considered, and often functional tests must be used (e.g. Northern blots or
qRT-PCR). qRT-PCR will often work fine with partially degraded samples .
18. tip #8
Storage of RNA
-For short-term storage, RNA should be stored at –20°C.
-For long-term storage, RNA should be stored at –80°C .
-Although RNA resuspended in water or buffer can be stored at –80°C, RNA
is most stable in an Ammonium Acetate /ethanol precipitation mixture kept at
–80°C .
-Aliquot your RNA into several tubes to prevent RNA damage from successive
freeze-thaw events and to prevent accidental RNase contamination.
20. tip #9
Integrate into workflow
-Regardless of the quantity and quality of RNA obtained, the downstream
results can be critically impacted by the specific reagents used.
-Life Technologies offers the best-in-class, validated reverse transcriptase
enzymes and reagents for real-time PCR.
-By using SuperScript® VILO™ cDNA Synthesis Kit with high quality isolated
RNA, your real-time PCR data will be the best it can be.
22. tip #10
Experimental design
-Always consider the overall experimental design to ensure validity.
-Run ≥3 biological replicates with technical replicates of the analytical
process. Can be reduced if the biological specimens are in an ordered series
(e.g. dosing levels or time points), where statistical vigor can be obtained by
the modeling of the response .
-Carefully matched controls provide a baseline that may change between
experiments, providing a more robust output, and helping to differentiate noise
from the interesting data.