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Validation of extracellular miRNA quantification in blood samples using RT-qPCR.

Author(s): Fauth M, Hegewald AB, Schmitz L, Krone DJ, Saul MJ

Publication: FASEB Bioadv, 2019, Vol. 1, Page 481-492

PubMed ID: 32123845 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of anticoagulant type, use of serum versus plasma, extraction method used, frozen storage of plasma, extracted RNA and cDNA, post-thaw mixing, and choice of real-time PCR normalizer on the quantification of four miRNAs in plasma and serum. The authors also determined acceptance criteria for quantification of miRNA in plasma. Blood was collected from a volunteer into Sarstedt S-Monovette K₃EDTA, sodium citrate, and lithium heparin plasma tubes and Sarstedt S-Monovette Serum-Gel Tubes and inverted three times before being placed on ice. Plasma and serum were obtained by centrifugation at 4°C for 20 minutes at 2000 x g and 2500 x g, respectively, aliquoted, and then stored at -80°C. Plasma and serum aliquots were thoroughly mixed before use unless otherwise specified. To test the effect of extraction method, RNA was extracted from EDTA plasma spiked with miR-146a-5p, miR-155-5p, miR-382-5p, and miR-451a using a phenol/guanidinium thiocyanate method in conjunction with Heavy Phase Lock Gel Tubes and using the miRNeasy Mini Kit. To determine the effect of tube type, plasma and serum were spiked with miR-146a-5p, miR-155-5p, miR-382-5p, and miR-451a before extraction using the phenol/GTC method. Extracted miRNAs were stored at -80°C before reverse transcription using the miScript II RT Kit and amplification using the miScript SYBRR Green PCR Assay. To test the stability of miRNA, plasma and isolated miRNAs were stored at -80°C and reverse-transcribed cDNA was stored at -20°C for 0, 1, 7, 30, and 120 days before quantification of miR-155-5p and miR-451a.

   Summary of Findings:

   Recovery of exogenous miR-146a-5p, miR-155-5p, miR-382-5p, and miR-451a was 44-54% higher using the phenol/guanidinium thiocyanate (GTC) method than the miRNeasy Kit (P<0.05, P<0.001, P<0.01, and P<0.05, respectively). As expected, quantification cycle values for each of the tested spiked-in miRNAs were higher for lithium heparin plasma than for other plasma or the serum but the difference was only significant for miR-451a (P<0.01, all). Although recovery of each of the miRNAs were comparable in K₃EDTA and sodium citrate plasma and serum and levels of miR-155-5p were not significantly affected by tube type, miR-146a-5p, miR-382-5p, and miR-451a levels were higher in K₃EDTA plasma than serum (P<0.0001, all), levels of miR-146a-5p and miR-382-5p were higher in sodium citrate plasma than serum (P<0.0001, both), and levels of miR-451a were higher in K₃EDTA plasma than sodium citrate plasma (P<0.0001). Precipitates were observed after thawing plasma slowly on ice. Further, decreased levels of miR-146a-5p and miR382-5p were found in thawed K₃EDTA and sodium citrate plasma when the specimen was separated rather than thoroughly mixed (P<0.05, all). However, no differences in miR-155-5p or miR-451a levels were observed in thoroughly mixed versus separated thawed plasma and none of the miRNAs in serum were affected by post-thaw mixing. Levels of miR-451a declined with frozen storage of extracted RNA at -80°C for ≥1 day (P<0.05) or frozen storage of plasma at -80°C for ≥ 30 days (P<0.01), but miR-155-5p was unaffected and miR-451a remained stable in cDNA stored at -20°C. A comparison of real-time normalization found superior performance (lowest CV) using cel-miR-39-3p for miR-146a-5p, miR-155-5p, and miR-451a but the CV of miR-382-5p was lowest when normalized with ath-miR-159a. Lastly, the inter- and intra-day precision of the CVs for each of the four miRNAs were <40% and <30%, respectively, at the lower limit of quantification (LLOQ) and <20% and <30%, respectively; at both higher concentrations. The intraday accuracy was outside the range of acceptance for all miRNA at LLOQ and for all miRNAs at the low concentration with the exception of miR-451a, but ranged from 2.72% (miR-155-5p) to 22.82% (miR-451a) at the middle concentration (25 or 250 pmol/L depending on miRNA). The authors defined acceptance criteria for miRNA in plasma as having a PCR efficiency of 100±10%, intra- and inter-day precision of <25% and <35%, respectively (extended to <30% and <40%, respectively at the LLOQ), and a calibration curve with an R² ≥0.98.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Anticoagulant	Potassium EDTA Sodium citrate Lithium heparin None
   Biospecimen Acquisition	Type of collection container/solution	Serum tube Plasma tube
   Storage	Storage conditions	As plasma As extracted RNA As cDNA
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-146a-5p miR-155-5p miR-382-5p miR-451a
   Analyte Extraction and Purification	Sample mixing	Thoroughly mixed Separated
   Analyte Extraction and Purification	Analyte isolation method	Phenol/guanidinium thiocyanate (GTC) method miRNeasy kit
   Real-time qRT-PCR Specific	Data handling	Normalized to cel-miR-39-3p Normalized to ath‐miR‐159a
   Storage	Storage duration	0 days 1 day 7 days 30 days 120 days

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