Total RNA was isolated from PG3-Oc-treated cells using the Quick-RNA mini prep kit (Zymo Research, Irvine, CA) according to the manufacturer's protocol. 500 ng of total RNA was used to generate cDNA using SuperScript III first-strand synthesis system with random primers (Invitrogen), following manufacturer's protocol. Real-time PCR was performed using POWER SYBR GREEN mast mix (Applied Biosystem) for DR5, p21, PUMA and GAPDH, and TaqMan primer-probes for detection of MYC mRNA levels on 7900HT Sequence Detection System (Applied Biosystem). PUMA primer (forward, 5’-GAC-GAC-CTC-AAC-GCA-CAG-TA-3’; reverse, 5’-AGG-AGT-CCC-ATG-ATG-AGA-TTG-T-3’), DR5 primer (forward, 5-ACAGTTGCAGCCGTAGTCTTG-3’,; 5’-CCAGGTCGTTGTGAGCTTCT-3), GAPDH primer (forward, 5′-TCG ACA GTC AGC CGC ATC TTC TTT-3′; reverse, 5′-ACC AAA TCC GTT GAC TCC GAC CTT-3′), Taq Prob IDs for MYC (HS 00153408) and GAPDH (HS 99999905). ∆∆Ct method was used to analyze and report fold-changes of the indicated genes.

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

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.