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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. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
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Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA
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QsRNA-seq: A protocol for generating libraries for high-throughput sequencing of small RNAs.

Alla Fishman1, Ayelet T Lamm1

  • 1Faculty of Biology, Technion-Israel Institute of Technology, Technion City, Haifa 32000, Israel.

Bio-Protocol
|March 3, 2021
PubMed
Summary

Researchers developed QsRNA-seq, a fast, gel-free method for preparing small RNA (sRNA) libraries for high-throughput sequencing. This technique improves accuracy and efficiency for sRNA expression profiling in research and clinical settings.

Keywords:
Gene expressionHigh-throughput sequencingSize-selectionSmall RNAUMImiRNApiRNAsiRNA

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Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing
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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Small RNAs (sRNAs) are crucial non-coding RNA molecules regulating cellular processes.
  • sRNAs are promising clinical biomarkers, driving demand for expression profiling.
  • Current sRNA library preparation for high-throughput sequencing (HTS) is challenging due to small molecule size.

Purpose of the Study:

  • To develop an efficient, gel-free method for small RNA library preparation for HTS.
  • To overcome the limitations of existing PAGE-based and gel-free protocols.
  • To enable high-depth and accurate sRNA expression profiling.

Main Methods:

  • Modified Solid Phase Reversible Immobilization (SPRI) for size selection of nucleic acids <100 nt with 20 nt length differences.
  • Developed QsRNA-seq, a gel-free protocol for sRNA library preparation.
  • Incorporated Unique Molecular Identifiers (UMIs) to reduce bias and quantify expression.

Main Results:

  • QsRNA-seq is a fast, easy-to-perform, and automatable protocol.
  • The method generates very clean libraries, yielding high-depth expression data.
  • QsRNA-seq effectively reduces library preparation biases and improves quantification accuracy.

Conclusions:

  • QsRNA-seq offers a superior solution for small RNA expression profiling.
  • The protocol meets the increasing demands for both research and clinical applications.
  • This method enhances the utility of sRNAs as biomarkers.