Absolute qpcr sybr low rox

LCLs were treated with either 100 μg/ml Cycloheximide (Sigma) or vehicle alone for 4 h and total RNA extracted with RNAeasy Kit (Qiagen) essentially as described by the manufacturer but with an additional 15 min DNAse I (Promega) digest of the samples on the column. 1 μg total RNA was annealed to oligodT(15) primer and reverse transcribed using M-MuLV RT (NEB) for 2 h at 42°C. After RNAse A digest, the cDNA was purified using PCR purification kit (Qiagen) and 1/40 of the eluate used per reaction. Three replicate qPCR reactions using ABsolute qPCR SYBR Low ROX (Thermo) were performed per experiment in a MX3005P (Agilent) thermocycler and analysed using MxPro software (Agilent). The fold change was calculated from ΔCt values relative to actin and ΔΔCt values relative to WT untreated for three independent experiments. Primers were:
XRCC1 exon10 forward: CAACACCCCCAAGTACAGC
XRCC1 exon 10 reverse: AGTCCAGCACCCACTCCTTAC
XRCC1 exon11 forward: TCCAGCAGTGAGGAGGATG
XRCC1 intron11 reverse: AGGCAAGAGTGGGAAGTTTG
XRCC1 exon 12 reverse: AGTGGGCTTGGTTTTGGTC
Actin forward: CTCGTCATACTCCTGCTTGC
Actin reverse: GAAGTGTGACGTGGACATCC