Mature miRNA Profiling and Quantification

As we described previously 21 (link), the extracted total RNA including miRNAs (10 ng/µl concentration) was first reverse transcribed into first strand cDNA using the RT2- miRNA First Strand Kit following manufacturer's recommendations (SA Biosciences, Rockville, MD). One µl cDNA per well was then mixed with SYBR Green qPCR Master Mix and placed into a 96-well PCR-array plate containing a panel of 88 mature miRNAs sequences. The arrays also contain appropriate small nucleolar RNA sequences that are used as housekeeping assays and quality controls. One µl was used in a 12 µl final volume reaction for Real-time PCR analysis on an Applied Biosystems Step-One Plus Real Time PCR system. Relative amounts were calculated by the ΔΔ CT method. Samples without good RNA quality were excluded in the statistical analysis.