Thermoscript rt

cDNA was prepared from total RNA (2.5 μg) using Thermoscript RT (Invitrogen) according to the instructions of the manufacturer. The sequences of all primers for qRT-PCR analysis were designed using the Primer Express 3.0 software (Applied Biosystems). The primers were tested to determine the optimal primer concentrations and efficiency. Combinations of the 50 nM, 300 nM and 900 nM (final concentration) per primer pair were checked, and based on the dissociation curve the optimal primer concentration per primer pair was chosen. The primer sequences of the tested genes and the reference gene are listed in Additional file 3: Table S3. qPCR analysis was performed by using the ABI 7500 fast real-time PCR system (Applied Biosystems). The reactions consisted of 2 μl forward and reverse primers at optimal concentration, 20 ng cDNA sample, 10 μl ABI Fast SYBR Master Mix (Applied Biosystems), and water to a final volume of 20 μl. The cycling parameters were 95 °C for 20 s, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. A dissociation curve was generated to verify that a single product was amplified. Transcript levels were normalized against the histone H2B gene expression and quantified according to the formula 2 –(Ct gene X – Ct H2B) [29 (link)]. Two biological and three technical replicates were analyzed.

HIV-1 subtype characterization was performed by full-length genome sequencing of HIV-1 RNA extracted from plasma using the QIAamp Viral RNA Mini Kit. Complementary DNA (cDNA) was synthesized as the complete genome or as two half genomes overlapping by 1.5 kb, using ThermoScript RT (Invitrogen Corp., Carlsbad, CA) as instructed by the manufacturer. Either primer JL68R (5’-CTTCTTCCTGCCATAGGAGATGCCTAAG-3’) or UNINEF-7’ (5’-GCACTCAAGGCAAGCTTTATTGAGGCTT-3’) was used as the 3’ primer to synthesize cDNA. With near-endpoint dilution of cDNA template, a full genome nested PCR was performed. MSF12b/UNINEF-7’ and GAG763 (5’- TGACTAGCGGAGGCTAGAAGGAGAGA-3’)/ TATANEF (5’-GCAGCTGCTTATATGCAGGATCTGAGGG-3’) were the primers used for full genome amplification. PCR products were purified and sequenced by an ABI 3100 capillary sequencer. DNA sequences were assembled using Sequencher version 4.7 and aligned with reference strains from the Los Alamos HIV-1 Database to generate a multiple sequence alignment. The viral strains were preliminarily genotyped using the NCBI Genotyping tool [28 (link)].

Total RNA (5 μg) from PC12 cells, isolated using RNeasy Mini Kit (Qiagen; Hilden, Germany) in accordance to the manufacturer instructions and treated with RNase-free DNase I (Qiagen; Hilden, Germany), was reverse transcribed with ThermoScript RT (Invitrogen) and Oligo dT primers. Synthesized cDNA (25 ng) was then combined in a PCR reaction using the appropriate primers and probes. Quantitative real-time PCR for the expression of neuronal specific differentiation markers was performed using TaqMan® Gene expression Assays: Gap43(Rn01474579_m1), Elavl4(Rn01416883_m1), Map2(Rn00565046_m1), Tubb3(Rn01431594_m1), B2m(Rn00560865_m1), according to the manufacturer's instructions. Quantitative real-time PCR for the expression of BDNF was performed using SYBR Green PCR Master Mix (PE Applied Biosystems) with the following primers: BDNF-Forward 5′-TCA AGC TGG AAG CCT GAA TGA A-3′, BDNF-reverse 5′-CCC AGT CAG GTA ACC ACT AAC AC-3′, using B2m as gene housekeeping: B2m-forward 5′-CCC ACC CTC ATG GCT ACT TC-3′, B2m-reverse 5′-GAT GAA AAC CGC ACA CAG GC-3′. Amplification reactions was performed on an ABI Prism 7500 (PE Applied Biosystems) according to the manufacturer's instructions. Relative quantitative determination of target gene levels was done by comparing ΔCt.

Total cellular RNA was extracted using TRIzol Reagent according to the manufacturer’s protocol. qRT-PCR was used to confirm the expression levels of mRNAs. Total RNA (2 μg) was reversely transcribed using the ThermoScript RT (Invitrogen) and oligo deoxy-thymidine (dT) primers. Synthesized cDNA was combined in a qRT-PCR reaction using primers for the gene of interest (Table 1).
Real-time PCR was performed with an ABI 7500 Real-Time PCR System (Applied Biosystems) using probe, primer sets and SYBR Green chemistry. Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin were used for normalization in SYBR Green chemistry. mRNA quantification was performed using the comparative cycle threshold (CT) method (ΔΔCt). For the real-time PCR estimation of the mitochondrial DNA (mtDNA) copy number total DNA samples were prepared using the cell DNA Isolation Kit (#53100, Norgen Biotek Corp). The mtDNA copy number was estimated by amplifying a portion of the tRNAleu of the mtDNA and comparing it to the amplification profile of a nuclear single copy gene, GAPDH.