Taqman probes for qpcr

Total RNA was isolated from the cultured cells and tissue was powdered according to the protocol supplied with QIAzol reagent (RNeasy lipid tissue kit, QIAGEN Inc., Valencia, CA, USA) according to the manufacturer’s instructions. The RNA was then quantified using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE), and 4 μg of the sample was used as a template for cDNA synthesis by reverse transcription with a SuperScript reverse transcriptase III kit (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed in a solution (20 μL) containing 2 TaqMan universal PCR mixture (10 μL), 20X TaqMan expression assay mix (1 μL), cDNA (50 ng), and primers. qRT-PCR was performed using a 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA). The conditions for thermal cycling were as follows: initial denaturation for 5 minutes, followed by 45 cycles of amplification at 95 °C for 15 s, annealing at 60 °C for 30 s, and extension at 76 °C for 30 s. After the PCR was complete, the cycle threshold (CT) value of each gene was verified and analysed using ΔΔCT. TaqMan probes for qPCR (Applied Biosystems) were HSD11B1, KRT10, KRT1, Loricrin, and RPL13A, using probes Hs00194153_m1, Hs0016628 9_m1, Hs00196158_m1, Hs01894962_s1*, and Hs04194366_g1, respectively.

RNA from fixed cells was extracted with RecoverALL Total Nuclei Acid Isolation kit (AM1975; Thermo Fisher Scientific). RNA from the nonfixed samples was extracted with Trizol (15596026; Invitrogen). cDNA was synthesized from 1 µg RNA with qScript XLT cDNA Super Mix kit (95161-100; Quanta Biosciences). qPCR was performed using FastStart Universal Probe Master kit (04914058001; Roche) with 40 ng cDNA per reaction. Taqman probes for qPCR (Applied Biosystems) are as follows: FOXA3, Hs00270130_m1; FOXJ1, Hs00230964_m1; P63, Hs00978340_m1; GAPDH, Hs99999905_m1; and TRRAP, Hs00268883_m1.