title: Identification of extracellular miRNA in archived serum samples by next-generation sequencing from RNA extracted using multiple methods.
abstract: miRNAs act as important regulators of gene expression by promoting mRNA degradation or by attenuating protein translation. Since miRNAs are stably expressed in bodily fluids, there is growing interest in profiling these miRNAs, as it is minimally invasive and cost-effective as a diagnostic matrix. A technical hurdle in studying miRNA dynamics is the ability to reliably extract miRNA as small sample volumes and low RNA abundance create challenges for extraction and downstream applications. The purpose of this study was to develop a pipeline for the recovery of miRNA using small volumes of archived serum samples. The RNA was extracted employing several widely utilized RNA isolation kits/methods with and without addition of a carrier. The small RNA library preparation was carried out using Illumina TruSeq small RNA kit and sequencing was carried out using Illumina platform. A fraction of five microliters of total RNA was used for library preparation as quantification is below the detection limit. We were able to profile miRNA levels in serum from all the methods tested. We found out that addition of nucleic acid based carrier molecules had higher numbers of processed reads but it did not enhance the mapping of any miRBase annotated sequences. However, some of the extraction procedures offer certain advantages: RNA extracted by TRIzol seemed to align to the miRBase best; extractions using TRIzol with carrier yielded higher miRNA-to-small RNA ratios. Nuclease free glycogen can be carrier of choice for miRNA sequencing. Our findings illustrate that miRNA extraction and quantification is influenced by the choice of methodologies. Addition of nucleic acid- based carrier molecules during extraction procedure is not a good choice when assaying miRNA using sequencing. The careful selection of an extraction method permits the archived serum samples to become valuable resources for high-throughput applications.
authors: US Army Center for Environmental Health Research, 568 Doughten Drive, Fort Detrick, 21702-5010, MD, USA.
authors: Aarti
authors: A
authors: Gautam
authors: Advanced Biomedical Computing Center, Frederick National Laboratory for Cancer Research/Leidos-Biomedical Inc., Frederick, MD, 21702, USA.
authors: Raina
authors: R
authors: Kumar
authors: Advanced Biomedical Computing Center, Frederick National Laboratory for Cancer Research/Leidos-Biomedical Inc., Frederick, MD, 21702, USA.
authors: George
authors: G
authors: Dimitrov
authors: The Geneva Foundation, US Army Center for Environmental Health Research, Fort Detrick, MD, 21702, USA.
authors: Allison
authors: A
authors: Hoke
authors: US Army Center for Environmental Health Research, 568 Doughten Drive, Fort Detrick, 21702-5010, MD, USA.
authors: Rasha
authors: R
authors: Hammamieh
authors: US Army Center for Environmental Health Research, 568 Doughten Drive, Fort Detrick, 21702-5010, MD, USA. marti.jett-tilton.civ@mail.mil.
authors: Marti
authors: M
authors: Jett
mesh_terms: D001800:Blood Specimen Collection; D020869:Gene Expression Profiling / Q000379:methods; D015723:Gene Library; D006146:Guanidines; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000097:blood* / Q000302:isolation & purification* / Q000592:standards; D010636:Phenols; D011933:Reagent Kits, Diagnostic; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D006146:Guanidines; D035683:MicroRNAs; D010636:Phenols; D011933:Reagent Kits, Diagnostic; C411644:trizol
keywords: Data analysis; DoDSR; Next-generation sequencing; Serum; miRNA