Results were analyzed by the SDS RQ Manager 1.2 software (Applied Biosystems, Foster City, CA).The relative miRNA expression level in each group was normalized to the housekeeping gene RNU6B and given by 2-ΔΔCt method. The formulae for the relative quantification of each of the miRNA were as follows: (dCt of each miRNA) = (Ct of each miRNA) − (Ct of internal control), and (relative quantification of each miRNA) = 2−(dCt of each miRNA). The Ct value was read according to the number of cycles at which the fluorescence signal passes a manual threshold. The Ct value of 39 was used as a cut-off point for defining a miRNA as not expressed.
ID_REF = VALUE = normalized signal (against housekeeping genes RNU6B)
Reverse transcription for miRNA microarray was carried out using Megaplex Primer pools, Human Pools A v2.1and B v3.0 (Applied Biosystems, Foster City, CA). The cDNA product was used to perform miRNA Array.
Total RNA was extracted with miRNeasy Mini Kit followed by DNase I treatment.
The specimens (both tumour and adjacent normal) were processed immediately after the section and snap-frozen in liquid nitrogen for RNA extraction.
Fresh human colorectal tissues were used for RNA extraction.