A complete workflow for high-throughput isolation of serum miRNAs and downstream analysis by qRT-PCR : application for cancer research and biomarker discovery

Biomarkers have become invaluable clinical research tools for cancer detection. There is an urgent need for biomarkers that are more sensitive and specific predictors to help decrease complications and morbidity related to prostate biopsies, and circulating microRNA (miRNA) in blood could potentially be a solution. With this future goal in mind, we developed a complete workflow providing: (1) high-throughput isolation of total serum RNA using the Applied BiosystemsTM MagMaxTM mirVanaTM Total RNA Isolation Kit for the Thermo ScientificTM KingFisherTM Flex or Duo instrument, with (2) downstream analysis of miRNA profiles by qRT-PCR using the Applied BiosystemsTM TaqManTM Advanced miRNA cDNA Synthesis Kit and TaqManTM Advanced miRNA Assays. The effectiveness of this workflow is exemplified here by analysis of a panel of miRNAs derived from prostate cancer serum samples versus a normal sample control group. By following this fast and easy workflow that is suitable for serum and other body fluids, disease-specific RNA signatures can be identified and used as biomarkers. Introduction An ideal biomarker should be easily assayed with minimally invasive procedures but possess high sensitivity and specificity. Despite many candidate biomarkers researched and proposed in the literature, very few have been able to meet these needs and translate over to clinical use. In fact, there are no reliable biomarker options available for certain types of cancer and other diseases. This highlights the growing need for more in-depth biomarker research and methods more amenable to greater sample throughput. The use of one biomarker, prostate-specific antigen (PSA), has led to a dramatic increase in the diagnosis of prostate cancer—over 230,000 new cases in 2014 [1]. While it is a sensitive test, less than one-fourth of patients undergoing a prostate biopsy based on PSA testing are actually found to have the disease. The identification of more sensitive and specific biomarkers could be highly beneficial in clinical research and for potential future clinical utility. Circulating RNA, and in particular serum miRNA, could potentially fill this void in the future, providing biomarkers more sensitive to detection. The main advantages of miRNAs versus longer molecules (mRNA, lncRNA) include: (1) a smaller number of sequences (<2,500) and thus ease of analysis and (2) stability due to small size (~16–27 nt) and thus robustness of detection. A number of serum miRNAs have been reported in the literature as potentially promising biomarkers of prostate cancer [2-4]. Here we describe a robust high-throughput workflow that enables highly sensitive and reproducible processing of clinical research sample types, including serum, to screen for miRNA biomarkers. Materials and methods RNA isolation Total RNA (including miRNA) was isolated from 100 μL of serum from individuals with prostate cancer and from healthy individuals (in duplicate) using the MagMAX mirVana Total RNA Isolation Kit and the Thermo ScientificTM KingFisherTM Flex Magnetic Particle Processor with 96 Deep-Well Head. MagMAX mirVana Total RNA Isolation Kit TaqMan Advanced miRNA Assays Analysis of miRNA targets cDNA was generated using 2 μL of purifi ed total RNA with the TaqMan Advanced miRNA cDNA Synthesis kit. qPCR was then performed (in triplicate) for each sample using 2 μL of diluted cDNA, TaqMan Advanced miRNA Assays, and Applied BiosystemsTM TaqManTM Fast Advanced Master Mix under fast cycling conditions. Analysis of mRNA targets cDNA was generated using 10 μL of total RNA with the Applied BiosystemsTM High-Capacity cDNA Reverse Transcription Kit. qPCR was then performed (in triplicate) for each sample using 2 μL of cDNA, TaqManTM Assay specifi c for the reference mRNA target, and TaqMan Fast Advanced Master Mix under fast cycling conditions. Results We have successfully developed a robust highthroughput workfl ow that enables processing of clinical research sample types, in particular serum, to identify miRNA biomarkers using qRT-PCR methodology. The high-throughput isolation method (Figure 1, left) allows faster processing of samples by using MagMAXTM bead-based chemistry, with alternative upfront purifi cation steps to replace organic extraction and the ability to run up to 96 samples at a time with the KingFisher Flex or Duo instrument. For downstream analysis of recovered RNA by qRT-PCR, the TaqMan Advanced miRNA cDNA Synthesis Kit uses a universal RT primer–based system to simplify the workfl ow and provide fl exibility for content coverage (Figure 1, right). The included preamplifi cation (miR-Amp) step allows universal amplifi cation for greater sensitivity to low-copy targets. The magnetic bead–based MagMAX mirVana Total RNA Isolation Kit offers superior performance and high-throughput capabilities for the RNA purifi cation step. Figure 2 highlights performance of the MagMAX mirVana kit versus the AmbionTM mirVanaTM PARISTM Kit, which employs a fi lter-based organic extraction method widely used for isolating total RNA (including