Whole Transcript Amplification
Whole Transcript Amplification - Related Videos
09:44
Rapid Amplification of cDNA Ends
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2023
Source: Pablo Sanchez Bosch2, Sean Corcoran2 and Katja Brückner1,2,3 1Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research 2Department of Cell and Tissue Biology, 3Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA Rapid Amplification of cDNA Ends (RACE) is a technique that allows amplification of full-length cDNA from mRNA by extending to the 3’ or 5’ end, even without prior knowledge of the sequence (Frohman et al.,...
09:38
Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer
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Cited by 3 •
2018
The two different 3' rapid amplification of cDNA ends (3' RACE) protocols described here make use of two different DNA polymerases to map sequences that include a segment of the open reading frame (ORF), the stop codon, and the entire 3' UTR of a transcript using RNA obtained from different cancer cell lines.
10:12
Protein Misfolding Cyclic Amplification of Prions
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Cited by 7 •
2012
Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.
05:34
Detecting SARS-CoV-2 Virus by Reverse Transcription-Loop-Mediated Isothermal Amplification
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Cited by 1 •
2023
Here we provide a complete protocol to standardize and implement the method for detecting the SARS-CoV-2 virus in human samples by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method, done within 60 min, could be adapted to any laboratory or point-of-care at a low cost and using inexpensive equipment.
02:23
Master Transcription Regulators
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2020
Master transcription regulators are regulatory proteins that are predominantly responsible for regulating the expression of multiple genes. Often these genes work in concert to drive a complex process. Activation of a master transcription regulator can lead to a cascade of transcriptional activation necessary for that outcome. These regulators can directly bind to the regulatory sequences of the various genes involved, or they can indirectly regulate transcription by binding to regulatory...
FAQs
The process first uses reverse transcription to create complementary DNA copies of the RNA population. Amplification then increases the amount of this cDNA through in vitro transcription or PCR. For the resulting material to remain biologically informative, the relative representation of transcripts should be preserved as closely as possible, allowing measurements to reflect the starting sample rather than amplification alone.
Reverse transcription provides the connection between the original RNA population and the amplified material analyzed later. It converts RNA molecules into cDNA, which can then serve as the substrate for an amplification step. This staging is important because the method depends on generating enough cDNA from scarce input while retaining information about the transcripts present in the biological sample.
Amplifying the total amount of material is useful only if the resulting cDNA population still represents the original transcript mixture. Preserving relative transcript representation supports meaningful gene-expression profiling and transcriptome analysis, because differences among transcripts can remain interpretable. If representation changes substantially during processing, measured abundance may describe amplification effects rather than the cellular state under investigation.
In vitro transcription and PCR are alternative enzymatic amplification routes used after RNA has been converted into cDNA. Both serve the practical purpose of increasing the available material from a small sample, while the broader objective remains faithful analysis of the original transcript population. The selected route therefore forms part of the method’s amplification stage rather than replacing reverse transcription.
It is particularly valuable when a study contains only a small biological sample, such as a single cell or a limited engineered tissue specimen. By extending the amount of analyzable transcript material, the approach can support gene-expression profiling and characterization of cellular states. It also helps researchers evaluate biological designs when obtaining larger amounts of starting material is impractical.
A typical workflow begins with the available RNA population, followed by reverse transcription to generate cDNA. The cDNA is then amplified through in vitro transcription or PCR, with attention to preserving relative transcript representation. The amplified material can subsequently support transcriptome analysis, gene-expression profiling, biomarker discovery, or assessment of engineered cells and tissues.
The resulting material can support comparisons of gene-expression patterns and broader transcriptome characteristics in engineered biological systems. These measurements may help identify cellular states, discover candidate biomarkers, and evaluate whether an engineered tissue or cell design produces the intended biological response. Interpretation remains tied to how faithfully the amplification reflects the original RNA population.