Superscript 3 platinum sybr green one step rtqpcr kit with rox

RNA was isolated from soleus and EDL muscles using TRIzol (Invitrogen, USA). Reverse transcription into cDNA was performed using 2 µg of total cellular RNA, 20 pmol oligo(dT) primer (Invitrogen), and reverse transcriptase (Advantage ImProm-II; Promega, USA). Real-time polymerase chain reaction (PCR) was carried out on a sequence detection system (Model ABI-7000; Applied Biosystems, USA), using the SuperScript III Platinum SYBR Green One-Step RTqPCR Kit with ROX (Invitrogen) with the primer of interest. Transcript levels of target gene were normalized with gapdh. Relative fold change in expression was calculated using the delta delta cycle threshold (ΔΔCT) method.

HIV-1 RNA was measured by reverse transcriptase quantitative PCR (RT-qPCR). Total RNA was isolated from SHIV-infected immortalized Ptm cells using the miRNeasy Mini Kit (Qiagen). For each reaction, 50 ng of total RNA, measured by Nanodrop spectrophotometer was amplified using the Superscript III Platinum SYBR Green One-Step RT-qPCR kit with ROX (Invitrogen). Primers 5’- AGGGACTTGGCAAATGGATTGTAC-3’ and 5’ GTGTAATAGGCCATCTGCCTGCC-3’ were used to amplify gag from unspliced RNA. To amplify splice vpu/env mRNA, the forward primer 5’-AGGAACCAACCACGACGGAGTGCTC -3’, which binds upstream of the splice donor site in the 5’ LTR, and reverse primer 5’-CATTGCCACTGTCTTCTGCTCTTTC-3’, which binds downstream of the Vpu start codon, were used. To amplify macaque β-actin mRNA, primers 5’-CAACCGCGAGAAGATGACCCAGATCATG-3’ and 5’-AGGATGGCATGGGGGAGGGCATAC-3’ were used. Relative levels of HIV-1 env mRNA for each virus was determined using the following equation: 2^-ΔΔ = [(C_Tenv—C_Tbeta actin)]—[(C_Tgenomic—C_Tbeta actin)] [43 (link)].