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Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA
Published on: October 27, 2011
Highly consistent, fully representative mRNA-Seq libraries from ten nanograms of total RNA
Srikumar Sengupta1, Victor Ruotti, Jennifer Bolin
1Morgridge Institute for Research, Madison, WI 53706, USA.
Biotechniques
|December 15, 2010
Summary
This study introduces a new RNA amplification protocol for gene expression analysis. The method requires minimal RNA input and generates high-quality sequencing libraries with reduced bias.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Standard Illumina sequencing library preparation for mRNA-Seq demands substantial RNA quantities (micrograms) or PCR amplification.
- Existing methods can introduce bias, affecting gene expression analysis accuracy.
Purpose of the Study:
- To develop a novel, low-input RNA amplification protocol for mRNA-Seq library preparation.
- To generate directional, whole-transcript Illumina libraries with minimal bias and high consistency.
Main Methods:
- Utilized T7 linear RNA amplification for library preparation.
- Tested the protocol with as little as 10 ng of total RNA.
Main Results:
- The T7 linear amplification protocol requires only 10 ng of total RNA, significantly reducing input requirements.
- The method generates directional, fully representative, whole-transcript mRNA-Seq libraries.
- High consistency was observed across a wide range of input RNA quantities (over three orders of magnitude).
- The protocol does not introduce significant bias into the sequencing data.
Conclusions:
- This T7 linear amplification method offers a low-input, unbiased, and consistent approach for mRNA-Seq library preparation.
- The protocol is suitable for gene expression analysis, especially when RNA is limited.
