Mx3005p detection system

Gene expression was quantified by SYBR Green real time-PCR using the MX3005P Detection System (Agilent Technologies, Böblingen, Germany). Primers are listed in the Supplementary table 1. Samples without enzyme in the reverse transcription reaction (Non-RT controls) were used as negative controls. Unspecific signals caused by primer dimers were excluded by non-template controls and by dissociation curve analysis^{62 (link)}. β-Actin was used to normalize for the amounts of complementary DNA within each sample.

Expression levels of HIV LTR were analyzed using real-time, quantitative PCR. Reverse transcription was performed with the 2720 Thermal cycler (Applied Biosystems) using aliquots of total RNA extracted from samples. The cDNA samples were diluted to 20 ng/ul and gene-specific primers were used: forward and reverse LTR HIV-1 primers and Taqman LTR-HIV (Cat. #VC00021 and VC00023, Sigma–Aldrich). To normalize the fold changes in gene expression, two internal control genes were used, GAPDH Taqman primer (Cat. #4331182, Life technologies) and 18S rRNA (Cat. #4333760F, Applied Biosystems). Fold changes were calculated based on uninfected control. All real-time PCR reactions were performed using the MX3005P detection system (Agilent Technologies) and the amplifications were done using the Brilliant II QPCR Master Mix (Cat. #600804, Agilent Technologies). The thermal cycling conditions were composed of an initial denaturation step at 95°C for 10 min, 45 cycles at 95°C for 30 s and 55°C for 1 min. The experiments were carried out in duplicates for each data point. The relative quantification in gene expression was determined using the 2^-ΔΔCt method (Livak and Schmittgen, 2001 (link)).

Total RNA was extracted as described above, and cDNA was reverse-transcribed from total RNA using FastQuant cDNA kit (Tiangen) following the manufacture' manual. RT-qPCR was performed using Mx3005P detection system (Agilent) at 95°C for 2 min, and then 40 cycles at 95°C for 20 s followed by 58°C for 20 s and 68°C for 20 s with ribosomal protein L27 gene (rpl27) as the endogenous control. cDNA equivalent to 20 ng of total RNA, 0.5 µM primer pairs and SYBR Green Real Master Mix (Tiangen) were used in each reaction. The 2^−ΔΔCt method was applied to analyze relative gene expression levels [20] . Primers used in RT-qPCR were given in Table S1.