Quantitative Analysis of mRNA and microRNA

For mRNA analysis, total RNA was extracted using the TRIzol (Life Techologies) reagent and retro-transcribed with the GoScript Reverse Transcription System (Promega) using oligo (dT) and random primers. qPCR analysis was performed with specific primers (Table 8) and analyzed as previously described [30 (link)]. qPCR analysis was performed using a 7500 Fast Real-Time PCR System (Applied Biosystems). Ribosomal protein L31 (rp-L31) was used as a reference gene to normalize quantitation of target genes for differences in the amount of total RNA in each sample.
microRNA levels were analyzed from total RNA using the following TaqMan MicroRNA Assays: miR-200a ID:000502, miR-200b ID:002251, miR-200c ID:002300 and RNU24 ID:001001(Applied Biosystems, Thermo Scientific). Relative expression was calculated using the comparative Ct method. The small nucleolar RNA U24 (RNU24) was used as a reference gene.
Both for mRNA and microRNA analysis, total RNA of HCC827 and HCC4006 parental cell lines were used as calibrator samples, relative to which differences in the RNA amount of resistant cell lines have been calculated. The data were analyzed using SDS (Ver. 1.4) software (Applied Biosystems).