Abi 9700 sequence detection system

RNA was isolated with Trizol reagent. 1 μg of total RNA was reverse transcribed to generated cDNA using the iScript cDNA synthesis kit. As a negative control parallel samples were run without reverse transcriptase. Non-quantitative PCR amplification was then performed to identify 100 bp amplicons with E6 and 36B4 primers as previously described [64 (link)]. For real-time RT-PCR, RNA was isolated and reverse transcribed as above and quantitative real-time PCR was performed using an ABI 9700 sequence detection system (Applied Biosystems, Foster City, CA). Amplification was carried out using TaqMan primer/probes: HPRT (Hs01003267_m1), GAPDH (Hs02758991_m1), BRCA1 (Hs01556193_m1 and Hs01556189_m1) and BRCA2 (Hs00609073_m1 and H201037421_m1) according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). Reactions were performed in triplicate in a 25 μL volume, with the following cycle parameters: enzyme activation (10 minutes at 95°C), followed by 40 cycles (each cycle consisting of 15 seconds at 95°C and 1 min at 60°C). Data analysis was performed using the comparative threshold cycle method (Applied Biosystems, Foster City, CA) to determine expression levels.

mRNA was TRIzol (Ambion) extracted from ~70% confluent HFK, SiHa, and Hela cells, before purification and concentration (Zymo Research). cDNA was synthesized with the iScript cDNA synthesis kit (Bio-Rad). RT-PCR was then performed using an ABI 9700 sequence detection system (Applied Biosystems). The following TaqMan primer/probes (ThermoFisher) were used: ACTB (Hs01060665_g1), PCNA (Hs00427214_g1), RPA70 (Hs00161419_m1), POLK (Hs00211965_m1), POLH (Hs00197814_m1), UBE2B (Hs00163311_m1), RAD18 (Hs00892551_m1), TOPBP1 (Hs00199775_m1), REV3L (Hs00161301_m1), REV1 (Hs01019768_m1), POLI (Hs00969214_m1).