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Isolation of Small Noncoding RNAs from Human Serum
Published on: June 19, 2014
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Comprehensive evaluation of extracellular small RNA isolation methods from serum in high throughput sequencing
Yan Guo1, Kasey Vickers2, Yanhua Xiong3
1Department of Cancer Biology, Vanderbilt University, Nashville, TN, USA.
BMC Genomics
|January 8, 2017
Summary
Comparing RNA isolation kits for circulating extracellular small RNAs (ex-sRNAs) reveals distinct performance profiles. The Ambion TRIzol method showed superior performance in small RNA sequencing (sRNAseq) yield and mapping efficiency for microRNAs (miRNAs), transfer RNA-derived small RNAs (tDRs), and other miscellaneous sRNAs (osRNAs).
Area of Science:
- Biochemistry
- Molecular Biology
- Genomics
Background:
- Circulating cell-free DNA and RNA fractions in blood are key analytes for disease diagnosis and staging.
- Small non-coding RNAs (sRNAs), including microRNAs (miRNAs), their isoforms (isomiRs), and transfer RNA-derived small RNAs (tDRs), show significant promise as disease biomarkers.
Purpose of the Study:
- To evaluate and compare the performance of five different commercial kits for isolating circulating extracellular small RNAs (ex-sRNAs).
- To identify the optimal method for small RNA sequencing (sRNAseq) library preparation based on yield and mapping efficiency.
Main Methods:
- Five kits were used to isolate ex-sRNAs from a single healthy individual: Qiagen Circulating Nucleic Acid Kit, ThermoFisher Scientific Ambion TRIzol LS Reagent, Qiagen miRNEasy, QiaSymphony RNA extraction kit, and Exiqon MiRCURY RNA Isolation Kit.
- Each isolation was performed in quadruplicate, generating 20 sRNAseq libraries.
- Libraries were sequenced and analyzed using a bioinformatics approach to compare RNA yield and mapping rates for different RNA classes.
Main Results:
- The Qiagen Circulating Nucleic Acid Kit exhibited high variability for miRNA isolation but low variability for other RNA classes (isomiRs, tDRs, osRNAs).
- ThermoFisher Scientific Ambion TRIzol LS Reagent demonstrated the best overall performance, yielding the highest number of mapped reads for miRNAs (10%), tDRs (7.2%), and osRNAs (23.1%).
- Other kits like QiaSymphony and miRNEasy showed intermediate performance, with QiaSymphony excelling in osRNA isolation and miRNEasy performing moderately for tDRs and miRNAs.
Conclusions:
- Each RNA isolation kit possesses unique performance characteristics, making them suitable for different downstream applications and sample processing needs.
- The choice of kit can significantly impact the yield and type of small RNAs detected, influencing biomarker discovery and diagnostic utility.
- Ambion TRIzol emerged as a top performer for comprehensive ex-sRNA profiling via sRNAseq in this comparative study.
