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Real Time RT-PCR

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Performance Comparison of Reverse Transcriptases for Single-Cell Studies.

Daniel Zucha^1,2, Peter Androvic^1,3, Mikael Kubista^1,4

¹Laboratory of Gene Expression, Institute of Biotechnology CAS, Vestec, Czech Republic.

Clinical Chemistry

|November 9, 2019

Summary

This summary is machine-generated.

This study compared 11 reverse transcriptases (RTases) for single-cell RNA quantification. Maxima H- and SuperScript IV demonstrated superior performance in capturing rare transcripts and improving experimental sensitivity and resolution.

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Area of Science:

Molecular Biology
Genomics
Biotechnology

Background:

Single-cell transcriptomic protocols heavily rely on reverse transcription (RT), a known source of variability, especially with low RNA amounts.
New reverse transcriptases (RTases) offer potential to reduce information loss, but their performance characteristics are not well-established.

Purpose of the Study:

To comprehensively compare the performance of 11 RTases under low RNA input conditions.
To identify optimal RTases for enhancing single-cell RNA quantification accuracy and sensitivity.

Main Methods:

Evaluated 11 RTases using quantitative reverse transcription PCR (RT-qPCR) on single-cell and 100-cell templates.
Utilized two priming strategies: conventional random hexamers/oligo(dT) and reduced oligo(dT) mimicking single-cell RNA sequencing (scRNA-seq) protocols.
Further tested the top two RTases in a high-throughput single-cell experiment.

Main Results:

All RTases showed high precision (R2 > 0.9445), but significant differences were observed in rare transcript capture (0%-90% positivity) and reaction yield (7.3%-137.9%).
Maxima H- and SuperScript IV were identified as the top-performing RTases.
Maxima H- demonstrated increased sensitivity and improved clustering resolution in single-cell experiments compared to SuperScript II.

Conclusions:

Identified Maxima H- and SuperScript IV as the best RTases for single-cell RNA quantification among 11 tested enzymes.
Provides a benchmark for selecting RTases to improve current single-cell quantification protocols and reduce variability.