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Related Concept Videos

RNA-seq03:21

RNA-seq

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 primer.
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Related Experiment Video

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Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos
08:37

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

Published on: October 9, 2020

Construction of small RNA cDNA libraries for deep sequencing.

Cheng Lu1, Blake C Meyers, Pamela J Green

  • 1Department of Plant and Soil Sciences, Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA.

Methods (San Diego, Calif.)
|September 25, 2007
PubMed
Summary

This study details methods for creating small RNA cDNA libraries for high-throughput sequencing. These techniques aid in identifying and quantifying microRNAs (miRNAs) and small interfering RNAs (siRNAs) for gene expression studies.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are critical regulators of gene expression in diverse organisms.
  • Traditional cloning methods have identified numerous miRNA and siRNA genes, but lack comprehensive quantitative data.
  • Advancements in sequencing technologies offer powerful tools for global analysis of small RNA populations.

Purpose of the Study:

  • To describe laboratory methods for constructing small RNA cDNA libraries.
  • To enable high-throughput sequencing of small RNAs for gene expression analysis.
  • To facilitate the identification and quantification of miRNAs and siRNAs.

Main Methods:

  • Construction of small RNA complementary DNA (cDNA) libraries.
  • Application of high-throughput sequencing technologies (e.g., MPSS, 454, SBS).
  • Method development for accurate small RNA profiling.

Main Results:

  • Established protocols for generating small RNA cDNA libraries suitable for deep sequencing.
  • Demonstrated the utility of these methods for identifying and quantifying small RNAs.
  • Provided a foundation for comprehensive gene expression studies using small RNA sequencing.

Conclusions:

  • The described methods are effective for constructing small RNA cDNA libraries for high-throughput sequencing.
  • These techniques advance the global analysis and quantitative expression profiling of miRNAs and siRNAs.
  • This work supports further research into the regulatory roles of small RNAs in biological systems.