M mlv reverse transcriptase

Total RNA was extracted using the GenElute^TM Mammalian Total RNA Miniprep kit (Sigma-Aldrich), and RNA concentration and purity were determined spectrophotometrically. For all samples, equal amounts of RNA were reverse transcribed into cDNA using M-MLV reverse transcriptase (Top-Bio, Prague, Czech Republic) and oligo-dT priming (Qiagen Inc., Valencia, CA, USA). Quantitative PCR was performed in 10 µL reaction volumes using the KAPA SYBR^® FAST qPCR Kit (Kapa Biosystems, Wilmington, MA, USA) and analyzed using the 7500 Fast Real-Time PCR System and 7500 Software v. 2.0.6 (both Life Technologies, Carlsbad, CA, USA). Technical triplicates were analyzed for each of the three biological replicates and relative gene expression levels were determined using the 2^−∆∆CT method [37 (link)]. The housekeeping gene HSP90AB1 was used as an endogenous reference control. The primer sequences used in this study are summarized in Table 3. NANOG primers were designed as previously published [38 (link)].

The relative expression levels of selected genes were studied using RT-qPCR. Total RNA was extracted using the GenElute™ Mammalian Total RNA Miniprep kit (Sigma-Aldrich), and RNA concentration and purity were determined spectrophotometrically. For all samples, equal amounts of RNA were reverse transcribed into cDNA using M-MLV reverse transcriptase (Top-Bio, Prague, Czech Republic). RT-qPCR was carried out in 10 μL reaction volumes using the KAPA SYBR^® FAST qPCR Kit (Kapa Biosystems, Wilmington, MA, USA) and analyzed using the 7500 Fast Real-Time PCR System and 7500 Software v. 2.0.6 (both Life Technologies, Carlsbad, CA, USA). Changes in the transcript levels were determined using the 2^−ΔΔCT method [33 (link)]. The housekeeping gene HSP90AB1 was used as an endogenous reference control. The primers used in this study are listed in Table 3.

The relative expression levels of genes of interest were studied using RT-PCR, and total RNA for this purpose was extracted using the GenElute™ Mammalian Total RNA Miniprep kit (Sigma-Aldrich). RNA concentration and purity were determined spectrophotometrically, and equal amounts of RNA were reverse transcribed into cDNA using M-MLV reverse transcriptase (Top-Bio, Prague, Czech Republic). RT-PCR was carried out in 20-μL reaction volumes using the GeneQ thermal cycler (BIOER). To ensure that the amplification does not reach a saturation plateau, the suitable number of cycles for analysis of each gene in question was selected on the basis of comparison of RT-PCR products after 25, 30, 35 and 40 cycles of reaction (S1 Fig). According to this initial analysis, expression level of each gene was semi-quantified after the 30 cycles of reaction, i.e. in the exponential phase of amplification. The products were subsequently separated with 1% agarose gel electrophoresis and visualized in a UV transilluminator (UVITEC Cambridge). Visualized transcript bands were quantified using ImageJ. The reference genes HSP90AB1 and GAPDH were used as an endogenous control. Data were analyzed using one-sample T-test (two-tailed); p< 0.05 was considered statistically significant. The primers used in this study are listed in Table 2.