Total RNA was extracted from samples using the RNeasy Mini Kit (Qiagen) and reverse-transcribed using the MMLV RT kit (Evrogen, Moscow, Russia). Oligonucleotide primers (Supplementary Table ) were ordered from Evrogen. Real-time PCR analysis was performed in triplicates using SYBR green qPCRmix-HS with ROX (Evrogen) with a StepOnePlus Real-Time PCR System (Applied Biosystems). Mean C t values for Wt1 (for SC-specific genes) and Hprt (for Acta2, Snai1 and Twist1) were used to calculate ΔC t values for each sample.
Wt1 is considered to be a stable SC marker and, according to our immunofluorescent data, it is continuously expressed in SCs throughout culture time. The melting curves for Wt1, Hprt. and genes analyzed gave only a single, unique peak for each primer set. ΔΔC t values for each sample were calculated by subtracting the mean ΔC t of the 34_5 TZ culture on day 2, except Snai1 and Twist1 for which the mean ΔC t of cultured mouse embryonic fibroblasts (MEF) was used. Relative quantification of RNA (RQ) was calculated using the 2 -ΔΔCt method (Livak & Schmittgen 2001).
Wt1 is considered to be a stable SC marker and, according to our immunofluorescent data, it is continuously expressed in SCs throughout culture time. The melting curves for Wt1, Hprt. and genes analyzed gave only a single, unique peak for each primer set. ΔΔC t values for each sample were calculated by subtracting the mean ΔC t of the 34_5 TZ culture on day 2, except Snai1 and Twist1 for which the mean ΔC t of cultured mouse embryonic fibroblasts (MEF) was used. Relative quantification of RNA (RQ) was calculated using the 2 -ΔΔCt method (Livak & Schmittgen 2001).