Custom openarray mirna rt pool

For cell line experiments, high throughput QuantStudio™ 12K Flex OpenArray^® PCR custom made plates were used for miRNA profiling. These arrays were comprised of a panel of 112 miRNA probes (miRBase version 22 miRNA names, seed sequences, and miRBase accession numbers are in Supplementary File S11) that were selected based upon their abundance in OAC patient samples from our previous study on serum small EV associated miRNAs [30 (link)]. For each sample, 3.35 μL of RNA, equivalent to 100 ng of RNA, was reverse transcribed using a matching Custom OpenArray^® miRNA RT pool (Life Technologies cat # A25630, Scoresby, Australia) and the TaqMan^® microRNA Reverse Transcription Kit (Life Technologies cat # 4366596). cDNA pre-amplifications were carried out with a matching Custom OpenArray^® PreAmp pool (Life Technologies cat # 4485255, Scoresby, Australia) and TaqMan PreAmp Master Mix (Life Technologies cat # 4488593, Scoresby, Australia) on 7.5 μL complementary DNA (cDNA) / sample for each pool. The pre-amplified products (4 μL per sample) were diluted at the recommended 1:40 dilution with 156 μL of RNase-free ultra pure water before mixing with TaqMan OpenArray Real-Time PCR Master Mix (Life Technologies cat # 4462164, Scoresby, Australia) and loading onto a 384-well TaqMan OpenArray loading plate. PCR runs were performed using a QuantStudio™ 12K Flex Real-Time PCR System.

High throughput QuantStudio™ 12 K Flex OpenArray^® PCR custom made plates were used for miRNA profiling. These arrays were comprised of a panel of 112 miRNA probes (Additional file 1) that were selected based upon their abundance in samples from our previous study on serum small extracellular vesicle associated miRNAs [22 (link)]. For each sample, 3.35 μl of RNA was reverse transcribed using a matching Custom OpenArray^® miRNA RT pool (Life Technologies cat # A25630) and the TaqMan^® microRNA Reverse Transcription Kit (Life Technologies cat # 4366596). cDNA Pre-amplifications were carried out with a matching Custom OpenArray^® PreAmp pool (Life Technologies cat # 4485255) and TaqMan PreAmp Master Mix (Life Technologies cat # 4488593) on 7.5 μl complementary DNA (cDNA)/sample for each pool. The pre-amplified products (4 μl per sample) were diluted at the recommended 1:40 dilution with 156 μl of RNase-free ultra pure water before mixing with TaqMan OpenArray Real-Time PCR Master Mix (Life Technologies cat # 4462164) and loading onto a 384-well TaqMan OpenArray loading plate. PCR runs were performed using a QuantStudio™ 12 K Flex Real-Time PCR System.