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RNA-seq03:21

RNA-seq

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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
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Real Time RT-PCR02:57

Real Time RT-PCR

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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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Related Experiment Video

Updated: Jan 17, 2026

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications
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2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

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Protocol for single-base quantification of RNA m5C by pyrosequencing.

Akin Cayir1, Yvonne Böttcher2

  • 1EpiGen, Medical Division, Akershus University Hospital, 1478 Lørenskog, Norway.

STAR Protocols
|September 20, 2025
PubMed
Summary
This summary is machine-generated.

We developed RNA-m5C-pyroseq, a novel method for precisely quantifying RNA methylation at specific sites. This technique analyzes 5-methylcytosine (m5C) modifications in RNA with single-nucleotide resolution.

Keywords:
Cell BiologyGeneticsGenomicsMolecular BiologySequencingSingle Cell

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Epigenetics

Background:

  • 5-methylcytosine (m5C) is a prevalent RNA modification.
  • m5C modifications occur in various coding and non-coding RNAs.
  • Accurate quantification of RNA methylation is crucial for understanding its biological roles.

Purpose of the Study:

  • To present a protocol for quantitative, single-nucleotide resolution analysis of RNA methylation.
  • To introduce the RNA-m5C-pyroseq method for m5C detection.
  • To provide detailed procedural steps for RNA methylation analysis.

Main Methods:

  • RNA isolation and preparation.
  • Bisulfite conversion of RNA.
  • Complementary DNA (cDNA) synthesis.
  • Polymerase Chain Reaction (PCR) amplification.
  • Pyrosequencing for methylation quantification.

Main Results:

  • The RNA-m5C-pyroseq protocol enables quantitative analysis of RNA methylation.
  • The method achieves single-nucleotide resolution for m5C detection.
  • Detailed procedures cover all steps from RNA isolation to pyrosequencing.

Conclusions:

  • RNA-m5C-pyroseq is an effective method for analyzing RNA methylation.
  • This protocol facilitates precise measurement of m5C modifications.
  • The technique supports further research into the functions of RNA methylation.