RNA was isolated from cells using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s instructions. One microgram of total RNA was used for complementary DNA (cDNA) synthesis. For standard mRNA analysis, oligo(dT)20 (Invitrogen) was added. For splice variant analysis, RNA was deoxyribonuclease (DNase)–treated (Sigma-Aldrich) before cDNA synthesis using random hexamers (Invitrogen). Moloney Murine Leukemia virus (M-MLV) reverse transcriptase (Promega) was used as per the manufacturer’s instructions. Quantitative reverse transcription PCR (qRT-PCR) was carried out in triplicate using the indicated primer pairs and the Brilliant III SYBR Green qPCR Master Mix (Stratagene) on an MX3005P (Agilent) qPCR instrument. Results were expressed as average (mean) fold change compared to control treatments using the ΔΔCt method from three biological repeat samples. Glyceraldehyde-phosphate dehydrogenase or actin primer sets were used as an internal calibrator. Error bars represent SE unless otherwise indicated.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.