Miscript pcr kit 2 rt kit

Total RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturer’s protocols. cDNAs were then synthesized using a Thermo-Script RT-PCR system (Invitrogen). mRNA amounts were measured by quantitative real-time PCR. The primers used here for human RhoGDIβ were 5’-ACC CGG CTC ACC CTG GTT TGT-3’ (Forward) and 5’-ACC CCA GTC CTG TAG GTG TGC TG-3’ (Reverse). Total microRNA was extracted using the miRNeasy Mini Kit (Qiagen). Analysis of miR-34a, -449, -383, -203a, -143, -7, and -219 expression was conducted using the miScript PCR Starter Kit and miScript PCR kit II RT Kit (Qiagen) following manufacturer protocols. U6 was always used as the endogenous normalizer. Initial activation was performed at 95℃ for 15 min, followed by 40 cycles of denaturation at 95℃ for 15 s, annealing at 55℃ for 30 s, and extension at 70℃ for 30 s. Cycle threshold (C_t) values were determined, and the relative expression of microRNAs calculated using values of 2^-△△Ct, as described earlier [48 (link), 49 (link)].

Total RNA from the cells was isolated using the trizol reagent. Total RNA (5 µg) was then used for reverse transcription with oligo dT primer through the SuperScript™ First-Strand Synthesis system IV (Invitrogen, Grand Island, NY). Specific primer pairs were designed to amplify human CDKN1B (forward: 5′-CAA GTA CGA GTG GCA AGA G-3′, reverse: 5′-ATG CGT GTC CTC AGA GTT AG -3′) and GAPDH (forward: 5′-AGA AGG CTG GGG CTC ATT TG-3′, reverse: 5′-AGG GGC CAT CCA CAG TCT TC-3′). Total miRNAs were extracted using the miRNeasy Mini Kit (Qiagen, Valencia, CA, USA). Total RNA (1.0 µg) was used for reverse transcription following the manufacturer’s instructions, and miRNA expression was determined by the Q6 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) using the miScript PCR Starter Kit and miScript PCR kit II RT Kit (Qiagen, Valencia, CA, USA). U6 was used as the endogenous normalizer. The primer for miR-190 (5′- TGA TAT GTT TGA TAT ATT AGG T-3′) was synthesized by Genewiz Biotechnology (South Plainfield, USA). The cycle threshold (CT) value was measured, and the relative expression of mRNA was calculated based on the value of 2^−ΔΔCT as described in our published studies [21 (link)].