Abstract
There are increasing reports of plasma miRNAs as biomarkers of human disease but few standards in methodologic reporting, leading to inconsistent data. We systematically reviewed plasma miRNA studies published between July 2013-June 2014 to assess methodology. Six parameters were investigated: time to plasma extraction, methods of RNA extraction, type of miRNA, quantification, cycle threshold (Ct) setting, and methods of statistical analysis. We compared these data with a proposed standard methodologic technique. Beginning with initial screening for 380 miRNAs using microfluidic array technology and validation in an additional cohort of patients, we compared 11 miRNAs that exhibited differential expression between 16 patients with benign colorectal neoplasms (advanced adenomas) and 16 patients without any neoplasm (controls). Plasma was isolated immediately, 12, 24, 48, or 72 h following phlebotomy. miRNA was extracted using two different techniques (Trizol LS with pre-amplification or modified miRNeasy). We performed Taqman-based RT-PCR assays for the 11 miRNAs with subsequent analyses using a variable Ct setting or a fixed Ct set at 0.01, 0.03, 0.05, or 0.5. Assays were performed in duplicate by two different operators. RNU6 was the internal reference. Systematic review yielded 74 manuscripts meeting inclusion criteria. One manuscript (1.4%) documented all 6 methodological parameters, while < 5% of studies listed Ct setting. In our proposed standard technique, plasma extraction ≤12 h provided consistent ΔCt. miRNeasy extraction yielded higher miRNA concentrations and fewer non-expressed miRNAs compared to Trizol LS (1/704 miRNAs [0.14%] vs 109/704 miRNAs [15%], not expressed, respectively). A fixed Ct bar setting of 0.03 yielded the most reproducible data, provided that <10% miRNA were non-expressed. There was no significant intra-operator variability. There was significant inter-operator variation using Trizol LS extraction, while this was negligible using modified miRNeasy. For standardized reporting, we recommend plasma extraction ≤ 12 h, using modified miRNeasy extraction and utilizing a 0.03 Ct.
Highlights
MicroRNAs are small 19–23 nucleotide noncoding ribonucleic acids (RNA) that bind to complementary sequences on the 3' untranslated region of target messenger RNAs [1]
When performing plasma extraction from whole blood at different time points, we found that 6 miRNAs showed no differences in ΔCT values
5 different sources of variation in experimental technique were examined with respect to their effect on miRNA expression data: 1) time of plasma extraction, 2) method of RNA extraction, 3) cycle threshold setting, 4) intra-operator variability and 5) inter-operator variability
Summary
MicroRNAs are small 19–23 nucleotide noncoding ribonucleic acids (RNA) that bind to complementary sequences on the 3' untranslated region of target messenger RNAs (mRNA) [1]. MiRNA profiles have, emerged as prospective biomarkers for cancer and many other human diseases [3–7]. MiRNA expression profiles have been shown to be unique to both the source material (i.e. plasma, tissue, etc.) and the disease process being investigated. This has led to a rapid proliferation of miRNA research. It is difficult to deduce the actual methods used for analysis This has led to the use of different extraction protocols, and various methods of quantification and statistical analysis, which, in turn, are a source of variability (Table 1). There is ongoing controversy over the optimal analytic methods for studies of miRNA in plasma[8]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
