Validation of extracellular miRNA quantification in blood samples using RT‐qPCR

Abstract

Extracellular microRNAs (miRs) have been proposed as important blood‐based biomarkers for several diseases. Contrary to proteins and other RNA classes, miRs are stable and easily detectable in body fluids. In this respect, miRs represent a perfect candidate for minimal invasive biomarkers which can hopefully become a complement for invasive histological examinations of tumor tissue. Despite the high number of miR biomarker studies, the specificity and reproducibility of these studies is missing. Therefore, the standardization of pre‐analytical and analytical methods is urgently needed. Here, we validated miR analysis for RNA isolation and miR quantification by quantitative polymerase chain reaction (RT‐qPCR) based on good laboratory practice (GLP). Validation was carried out exemplarily on four miRs, which had already been described as potential biomarkers in previous studies. As basis for RNA analysis using RT‐qPCR, the Minimum Information for Publication of Quantitative Real‐Time PCR Experiments were applied and adapted on the analysis of circulating miRs from human plasma. In our study, we identified and solved several pitfalls from handling to normalization strategy in the analysis of extracellular miRs that lead to inconsistent and non‐repeatable data. Principles of GLP set a framework of experimental design, performance and monitoring to ensure high quality and reliable data. Within this study, we appointed first acceptance criteria for circulating miR quantification during validation which set standards for future miR quantification in blood samples.

Extracellular miR-574-5p Induces Osteoclast Differentiation via TLR 7/8 in Rheumatoid Arthritis

“…Residual DNA was removed by on-column DNAse digestion using RNase-Free DNase Set (Qiagen, Hilden, GER). Total RNA from purified sEV was isolated using the phenol/guanidinium thiocyanate (GTC)-based extraction method according to ( 18 ). 200 nM of a non-human synthetic cel-miR-39-3p (5′-UCACCGGGUGUAAAUCAGCUUG-3′, Sigma Aldrich, Darmstadt, GER) was added as internal standard to compensate for technical and methodical variations.…”

“…Intracellular and the extracellular miR quantification were performed using RT-qPCR analysis according to ( 14 , 18 ). qRT-PCR was performed using the following primer: for miR-16-5p (MS0031493), miR-146a-5p (MS00003535), miR-155-5p (MS00031486), miR-574-5p (MS00043617, all from Qiagen, Hilden, GER).…”

Standardisierte mikroRNA-Analytik — die große Chance für Biomarkerstudien?

“…2C). Alternativ kann durch eine Validierung nachgewiesen werden, dass die Verwendung verschiedener Antikoagulantien oder vorhandene Proteinaggregate die miR-Analyse nicht beeinfl usst [5].…”

“…Die widerspr ü c hlichen Ergebnisse sind weitgehend auf die Heterogenität der präanaly-tischen und analytischen Schritte zurückzuführen. Um valide miR-Biomarker zu entwickeln, ist daher eine Validierung und Standardisierung der Methoden notwendig [5].…”

“…Wir haben dieses Prinzip auf die Analyse extrazellulärer miRs angewandt und Akzeptanzkriterien für ihre Quantifi zierung in humanem Plasma durch RT-qPCR definiert (Abb. 2C, [5]). Abhängig vo n der untersuchten miR be obachteten wir signifi kante Unterschiede in der Quantifi zierung, die von Faktoren wie hemmen oder die Freisetzung von miRs aus Erythrozyten fördern [7].…”