Total RNA containing both miRNA and mRNA was extracted using the Qiagen miRNeasy kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s protocol. Only total RNA of highest quality and integrity was subjected to further processing after purification as defined by an absorption ratio 260/280>1.8 by spectrophotometry on the NanoDrop 1000 (NanoDrop, Wilmington, DE) and a RIN value >8.0 via electrophoretic analysis on the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA).
Label
biotin
Label protocol
RNA was quantified with the Nanodrop (Thermo Scientific, IL). Reverse transcription was performed using iScript RT Supermix followed by PCR with SsoADV SYBR Green (Bio-Rad, CA) on an ABI StepOne Plus Real-Time PCR machine (Applied Biosystems, CA). Primers were designed using Primer Express software (Applied Biosystems, CA) and purchased from Life Technologies (NY) (Table 3).
Hybridization protocol
Fifteen micrograms of purified, amplified, biotin labeled cRNA was fragmented and hybridized on to Affymetrix Human Genome HGU133A 2.0 arrays (Affymetrix Corp. Santa Clara, CA) for 18 hours.
Scan protocol
Washing, staining and scanning of arrays was performed on the Affymetrix Fluidics Station 450 and Scanner 3000 immediately after completion of hybridization.
Data processing
Microarray data was processed using the Robust Multi-array Average by importing raw CEL files into Partek software (Partek Genomics Suite, St. Louis, MO) resulting in RMA background correction, log 2 transformation, quantile normalization, and median polish probeset summarization. HG-U133A_2.cdf HG-U133A_2.na32.annot
