TY - JOUR

T1 - miRNA Expression in Ovarian Cancer in Fresh Frozen, Formalin-fixed Paraffin-embedded and Plasma Samples

AU - Petersen, Patrick H.D.

AU - Lopacinska-Jørgensen, Joanna

AU - Oliveira, Douglas V.N.P.

AU - Høgdall, Claus K.

AU - Høgdall, Estrid V.

N1 - Funding Information: The Authors are grateful to the Danish Cancer Biobank (Bio-and GenomeBank, Denmark - RBGB) and the Danish Gynecologic Cancer Database (DGCD) for making specimens and data available for use in the present study. This work was founded by: The Mermaid Foundation, URL: http://www.mermaidprojektet.dk/ (PHDP, JLJ, CKH and EVH received the funding), Danish Cancer Research Foundation, URL: http://www.dansk-kraeftforsknings-fond.dk/ (EVH received the funding), and Herlev Hospital Research Council, URL: https://www.herlevhospital.dk/forskning/ (EVH received the funding). The Authors thank Ib Jarle Christensen for discussion of the statistical analyses performed in the study. Funding Information: The Authors are grateful to the Danish Cancer Biobank (Bio- and GenomeBank, Denmark - RBGB) and the Danish Gynecologic Cancer Database (DGCD) for making specimens and data available for use in the present study. This work was founded by: The Mermaid Foundation, URL: http://www.mermaidprojektet.dk/ (PHDP, JLJ, CKH and EVH received the funding), Danish Cancer Research Foundation, URL: http://www.dansk-kraeftforskningsfond.dk/ (EVH received the funding), and Herlev Hospital Research Council, URL: https://www.herlevhospital.dk/forskning/ (EVH received the funding). The Authors thank Ib Jarle Christensen for discussion of the statistical analyses performed in the study.

PY - 2022

Y1 - 2022

N2 - 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.

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.

KW - epithelial ovarian cancer

KW - miRNA

KW - normalization

KW - RT-qPCR

KW - spike-in controls

U2 - 10.21873/invivo.12869

DO - 10.21873/invivo.12869

M3 - Journal article

C2 - 35738639

AN - SCOPUS:85132683165

VL - 36

SP - 1591

EP - 1602

JO - In Vivo

JF - In Vivo

SN - 0258-851X

IS - 4

ER -