Superprep rt kit for qpcr

Cells were treated with doxycycline (Sigma-Aldrich, #D9891, 200 ng/ml) for 72 h. Total RNA was isolated using SuperPrep Cell Lysis kit (TOYOBO, #SCQ-101) as described by manufacturer and reverse transcribed with SuperPrep RT kit for qPCR (TOYOBO, #SCQ-101) for cDNA synthesis. Target genes were detected by real time PCR in CFX96 Touch™ according to manufacturer's protocol. Transcripts were quantified relative to the housekeeping gene, GAPDH. The probes used for this study were listed in Table S7.

The NMP-R mESCs were transfected with sgRNA expression vectors and treated as described above. Cells were then transferred onto 96-well plates, 6 h before RNA extraction. Total RNA was isolated from cells using a SuperPrep Cell Lysis Kit (Toyobo, Osaka, Japan) for quantitative PCR (qPCR) and for reverse transcription (RT)-PCR according to the manufacturer's protocol. Total RNA was converted to cDNA using a SuperPrep RT Kit for qPCR (Toyobo). qPCR was performed with the Stratagene Mx3000p (Agilent Technologies, Palo Alto, CA, USA) using the THUNDERBIRD SYBR qPCR Mix (Toyobo). The threshold cycle (Ct) value was normalized with the housekeeping gene Gapdh and the relative fold change was computed by the ΔΔCt method (22 (link)). Primer sequences are listed in Supplementary Table S2.