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RT-qPCR assay

SS Soichiro Sonoda
KY Koichiro Yoshimaru
HY Haruyoshi Yamaza
RY Ratih Yuniartha
TM Toshiharu Matsuura
EY Erika Yamauchi-Tomoda
SM Sara Murata
KN Kento Nishida
YO Yoshinao Oda
SO Shouichi Ohga
TT Tasturo Tajiri
TT Tomoaki Taguchi
TY Takayoshi Yamaza
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RNA samples were extracted from the cell and tissue samples using TRIzol reagent (Thermo Fisher Scientific), digested with DNase I (Promega), and purified using an RNeasy Mini Kit (Qiagen). cDNA was prepared from the purified total RNA by reverse transcription using a Revertra Ace qPCR kit (TOYOBO, Tokyo, Japan) according to the manufacturer’s instructions and used for RT-qPCR assays. Gene expression was analyzed by RT-qPCR using the cDNA mixed with EagleTaq Master Mix (Roche Applied Science, Babaria, Germany) and target TaqMan probes (Thermo Fisher Scientific; Additional file 1: Tables S3 and S4) with a Light Cycler 96 system real-time PCR cycler (Roche Applied Science). The PCR steps were as follows: preincubation 1 at 50 °C for 120 s, preincubation 2 at 95 °C for 600 s, two-step amplification at 95 °C for 15 s, and 60 °C for 60 s (45 cycles). Human and mouse 18S ribosomal RNA were used for normalization.

Copyright and License information: The Author(s) ©2021
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