RT Journal Article
SR Electronic
T1 miRNA Expression in Ovarian Cancer in Fresh Frozen, Formalin-fixed Paraffin-embedded and Plasma Samples
JF In Vivo
JO In Vivo
FD International Institute of Anticancer Research
SP 1591
OP 1602
DO 10.21873/invivo.12869
VO 36
IS 4
A1 PATRICK H.D. PETERSEN
A1 JOANNA LOPACINSKA-JØRGENSEN
A1 DOUGLAS V.N.P. OLIVEIRA
A1 CLAUS K. HØGDALL
A1 ESTRID V. HØGDALL
YR 2022
UL http://iv.iiarjournals.org/content/36/4/1591.abstract
AB Background/Aim: MicroRNAs (miRNAs) are small noncoding RNAs involved in gene expression regulation and have been investigated as potential biomarkers for various diseases, including ovarian cancer (OC). However, lack of standardized protocols regarding e.g., RNA isolation, cDNA synthesis, spike-in controls for experimental steps, and data normalization, impacts cross validation of results across research groups and hinders implementation of miRNAs as clinical biomarkers. Materials and Methods: RNA was isolated from matching fresh-frozen tissue (FF), formalin-fixed paraffin embedded (FFPE) tissue, and plasma samples from twenty women diagnosed with OC using three commercial kits: miRNeasy Tissue/Cells, miRNeasy FFPE, and miRNeasy Serum/Plasma (Qiagen, Copenhagen, Denmark). RNA isolation, cDNA synthesis, and PCR performance were tested using miRCURY LNA miRNA Quality Control PCR (QC) Panels (Qiagen). Finally, miRNA stability was assessed using five algorithms: BestKeeper, Normfinder, GeNorm, comparative delta-Ct and comprehensive ranking provided by a web-based RefFinder tool. Results: RNA from FF, FFPE and plasma was extracted using commercially available kits and the differences in yield and purity were examined. We developed a simple method for identifying and potentially excluding samples based on their crossing point values from RT-qPCR data, which could improve existing manufacture guidelines. Moreover, we discussed how assessment of miRNA stability differs between algorithms, possibly leading to inconsistent results. Conclusion: We present guidelines for RNA isolation, cDNA synthesis, and data normalization for successful miRNA expression profiling using RT-qPCR in corresponding biological OC specimens. We recommend QC panels in combination with spike-in controls and interplate controls to monitor process efficiencies.