Quantitative Analysis of miRNA and mRNA

RNA was isolated from cells using the miRCURY RNA Isolation Kit (Exiqon). cDNA was synthesized from small non-coding RNA using the miRNA RT assay (TaqMan). Expression levels of miRNA were measured on a 7900 fast RT-PCR system (Applied biosystems) in triplicates using 10 ng/µl cDNA and TaqMan probes specific for miR-138. U6 probe was used as an endogenous control. The ΔΔCt method was applied to determine the transcript abundance. For cDNA preparation from total mRNA, SuperScript III Reverse Transcriptase was used. Quantitative Real Time PCR (qRT-PCR) analyses were performed using primers that amplify a coding region of the TUSC2 gene. qRT-PCR was performed in triplicates with SYBR™ Green master mix (Applied Biosystems), 0.2 uM primers and 10 ng/µl cDNA. Adapted from Pascale et al.^{32 (link)}.