title: Comprehensive evaluation of extracellular small RNA isolation methods from serum in high throughput sequencing.
abstract: DNA and RNA fractions from whole blood, serum and plasma are increasingly popular analytes that are currently under investigation for their utility in the diagnosis and staging of disease. Small non-coding ribonucleic acids (sRNAs), specifically microRNAs (miRNAs) and their variant isoforms (isomiRs), and transfer RNA (tRNA)-derived small RNAs (tDRs) comprise a repertoire of molecules particularly promising in this regard.
  
  
  
  In this designed study, we compared the performance of various methods and kits for isolating circulating extracellular sRNAs (ex-sRNAs). ex-sRNAs from one healthy individual were isolated using five different isolation kits: Qiagen Circulating Nucleic Acid Kit, ThermoFisher Scientific Ambion TRIzol LS Reagent, Qiagen miRNEasy, QiaSymphony RNA extraction kit and the Exiqon MiRCURY RNA Isolation Kit. Each isolation method was repeated four times. A total of 20 small RNA sequencing (sRNAseq) libraries were constructed, sequenced and compared using a rigorous bioinformatics approach. The Circulating Nucleic Acid Kit had the greatest miRNA isolation variability, but had the lowest isolation variability for other RNA classes (isomiRs, tDRs, and other miscellaneous sRNAs (osRNA). However, the Circulating Nucleic Acid Kit consistently generated the fewest number of reads mapped to the genome, as compared to the best-performing method, Ambion TRIzol, which mapped 10% of the miRNAs, 7.2% of the tDRs and 23.1% of the osRNAs. The other methods performed intermediary, with QiaSymphony mapping 14% of the osRNAs, and miRNEasy mapping 4.6% of the tDRs and 2.9% of the miRNAs, achieving the second best kit performance rating overall.
  
  
  
  In summary, each isolation kit displayed different performance characteristics that could be construed as biased or advantageous, depending upon the downstream application and number of samples that require processing.
authors.affiliation: Department of Cancer Biology, Vanderbilt University, Nashville, TN, USA.
authors.forename: Yan
authors.identifier: 0000-0001-5252-3960
authors.initials: Y
authors.lastname: Guo
authors.affiliation: Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: Kasey
authors.initials: K
authors.lastname: Vickers
authors.affiliation: Department of Surgery, Vanderbilt University, MRBIV 8465A Langford Hall, 2213 Garland Ave, Nashville, TN, 37232, USA.
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Xiong
authors.affiliation: Department of Cancer Biology, Vanderbilt University, Nashville, TN, USA.
authors.forename: Shilin
authors.initials: S
authors.lastname: Zhao
authors.affiliation: Department of Cancer Biology, Vanderbilt University, Nashville, TN, USA.
authors.forename: Quanhu
authors.initials: Q
authors.lastname: Sheng
authors.affiliation: Department of Cancer Biology, Vanderbilt University, Nashville, TN, USA.
authors.forename: Pan
authors.initials: P
authors.lastname: Zhang
authors.affiliation: Center for Epigenetics, Van Andel Research Institute, Grand Rapids, MI, 49503, USA.
authors.forename: Wanding
authors.initials: W
authors.lastname: Zhou
authors.affiliation: Department of Surgery, Vanderbilt University, MRBIV 8465A Langford Hall, 2213 Garland Ave, Nashville, TN, 37232, USA. robb.flynn@vanderbilt.edu.
authors.forename: Charles R
authors.initials: CR
authors.lastname: Flynn
mesh_terms: D005110:Extracellular Space / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D058727:RNA, Small Untranslated / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification*; D017423:Sequence Analysis, RNA
chemical_list: D058727:RNA, Small Untranslated