Profiling Microarray-Based miRNA Expression

MicroRNA profiling was performed using the OpenArray platform from Thermo Fisher, as described previously^{66 (link)}. Briefly, the OpenArray reverse transcription reaction was performed according to the manufacturer’s protocol using 3 µl of total RNA pooled from 3 samples. Before loading onto the OpenArray, cDNA was pre-amplified following the manufacturer’s recommendation. Then, the pre-amplified product was diluted with 0.1X TE (1/40) and 22.5 µl of diluted pre-amplified product was added to the same volume of 2X Taqman OpenArray Real time PCR Master Mix (Cat No. 4462164, AB). Finally, the mix of Pre-Amp product and Master-Mix was loaded onto a 384-well OpenArray plate. OpenArray panels were automatically loaded by the OpenArray AccuFill System (Thermo Fisher Scientific, Waltham, Massachusetts) and run on a QuantStudio 12 K Flex Real-Time PCR system. Each panel enables the quantification of microRNA expression of three samples. 754 microRNAs were amplified from each sample together with 16 replicates of four internal controls (ath-miR159a (negative control), RNU48, RNU44 and U6 rRNA).