Itaq universal sybr green supermix

The expression levels of some genes in related signal pathways of TPCSs on ultra‐hydrogels activated by muscle contraction simulation were verified by a quantitative real‐time PCR, including itgb1, yap, taz, mkx, tnmd, co1a1 and col1a2. For detection of itgb1, yap and taz, at Day 4, the TPSCs cultured on the hydrogels of 0%, 20%, 50% were washed and broken for extracting the total RNA, with reverse‐transcribed into complementary DNA using a PrimeScript RT reagent kit. The expression level of mkx, tnmd, co1a1 and col1a2 were detected from the samples from each group collected at Day 14. qRT‐PCR was carried out by employing iTaq universal SYBR Green supermix (Applied Biosystems, USA) with the gene‐specific primers listed in Table S2, Supporting Information.

C. albicans pellets were collected (A₆₀₀ = 20) and RNA isolated using a Total RNA Mini Kit according to the manufacturer’s instructions. The concentration and purity of the isolated RNA was measured using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). In order to remove genomic DNA, samples were treated with DNAse I. The isolated RNA from all samples was brought to an equal concentration (14 ng/μL) and the cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit.
The reaction was carried out using specific primers for genes ACT1 (reference gene) and ERG11 as follows: ACT1F (5′-TCCAGCTTTCTACGTTTCCA-3′), ACT1R (5′-GTC AAGTCTCTACCAGCCAA-3′), ERG11F (5′-TTTGGTGGTGGTAGACATA-3′), ERG11R (5′-GAACTATAATCAGGGTCAGG-3′). RT-qPCR reaction was conducted using the iTaq Universal Sybr Green Supermix and the StepOnePlus Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The initial step of the thermal cycling program was performed at 95 °C for 10 min, followed by 40 cycles at 95 °C for 20 s, 45 °C for 20 s, and 72 °C for 30 s. The determination of gene expression levels was performed as described previously using the 2^–∆∆Ct method [39 (link)].