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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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Fast TV-PRO-seq: Accelerated and Streamlined Protocol for Timing RNA Polymerase Pausing.

Jie Zhang^1,2,3,4,5, Zhixian Liang^1,2,3, Mingxin Sun⁴

¹National Engineering Research Center for Healthcare Devices & Guangdong Provincial Key Laboratory of Medical Electronic Instruments and Materials, Institute of Biological and Medical Engineering, Guangdong Academy of Sciences, Guangzhou, China.

|July 31, 2025

Summary

This summary is machine-generated.

Fast TV-PRO-seq offers precise, genome-wide mapping of RNA polymerase II pausing times at single-nucleotide resolution. This optimized protocol significantly reduces experimental time and cell input, enhancing feasibility for studying gene expression dynamics.

Keywords:

Dynamic transcriptional regulation Nascent transcription Next-generation sequencing (NGS)PRO-seq RNA polymerase II pausing duration RNA polymerase pausing Single-base resolution

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Area of Science:

Molecular Biology
Genomics
Epigenetics

Background:

Transcriptional pausing is crucial for regulating gene expression during cellular processes.
Accurate measurement of pausing duration is essential but technically challenging.
Existing methods lack the resolution and efficiency needed for comprehensive analysis.

Purpose of the Study:

To introduce Fast TV-PRO-seq, an optimized protocol for precise genome-wide mapping of RNA polymerase II pausing times.
To overcome limitations in resolution and experimental efficiency of previous methods.
To enable detailed analysis of transcriptional pausing dynamics.

Main Methods:

Developed Fast TV-PRO-seq based on time-variant precision run-on sequencing (TV-PRO-seq).
Utilized sarkosyl-free biotin-NTP run-on with time gradients and on-bead enzymatic reactions.
Integrated bash scripts for streamlined workflows and simplified computational analysis.

Main Results:

Achieved single-nucleotide resolution mapping of RNA polymerase II pausing times genome-wide.
Reduced experimental duration from 4 days to 2 days.
Enabled experiments with reduced cell input (10^6-10^8 cells) and under sarkosyl-free conditions.

Conclusions:

Fast TV-PRO-seq provides a highly efficient and accurate method for studying transcriptional pausing.
The protocol enhances experimental feasibility and reproducibility.
This advancement facilitates deeper understanding of gene expression regulation.