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.
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.
Authors: Thorarinn Blondal; Søren Jensby Nielsen; Adam Baker; Ditte Andreasen; Peter Mouritzen; Maria Wrang Teilum; Ina K Dahlsveen Journal: Methods Date: 2012-10-02 Impact factor: 3.608
Authors: Joanna Lopacinska-Joergensen; Douglas V N P Oliveira; Claus K Hoegdall; Estrid V Hoegdall Journal: In Vivo Date: 2022 May-Jun Impact factor: 2.406