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Related Concept Videos

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. 
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Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific...
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Related Experiment Video

Updated: Sep 2, 2025

A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools
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Cloning and Sequencing Eukaryotic Small RNAs.

Olivia J Crocker1,2, Natalie A Trigg1,2, Colin C Conine1,2

  • 1Division of Neonatology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.

Current Protocols
|August 4, 2022
PubMed
Summary
This summary is machine-generated.

This study guides researchers on accurately cloning eukaryotic small RNAs. It addresses limitations in traditional methods for detecting modified small RNAs, improving abundance and diversity assessments.

Keywords:
RNA-seqRNAimicroRNAspiRNAssmall RNAtRNA-fragments

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Small RNAs regulate gene expression across eukaryotes.
  • Diverse small RNA classes exist, grouped by biogenesis and function.
  • Traditional cloning methods have limitations for detecting modified small RNAs.

Purpose of the Study:

  • To guide the design of small RNA cloning workflows.
  • To improve the accurate capture of specific small RNA classes.
  • To address limitations in current small RNA identification protocols.

Main Methods:

  • Review of molecular biology of small RNA identification.
  • Analysis of limitations in traditional small RNA cloning protocols.
  • Exploration of alternative approaches for specific small RNA enrichment.

Main Results:

  • Traditional protocols may fail to detect modified small RNAs.
  • Chemical modifications can impede adapter ligation and reverse transcription.
  • Inaccurate cloning leads to skewed representation of small RNA abundance and diversity.

Conclusions:

  • Accurate small RNA cloning is crucial for understanding gene regulation.
  • Alternative workflows are needed to capture modified small RNAs.
  • Improved methods enhance the assessment of small RNA diversity and abundance.