Quantstudio 6 flex pcr system

Total RNA was extracted from mouse spinal cord tissues by Tissue RNA Purification Kit (ESscience Biotech, China) and reversely transcribed into cDNA using Fast All-in-One RT Kit (ESscience Biotech, China). Then, RT-PCR was performed using cDNA as the template with Super SYBR Green qPCR Master Mix (ESscience Biotech, China) and QuantStudio 6 Flex PCR System (Applied Biosystems, United States). GAPDH was used as the reference control, and the results were analyzed using the 2^−ΔΔCT method. The primer sequences of target genes are shown in Table 1.

RNA isolation, reverse transcription and detection by RT-PCR in rats were performed as described previously (18,19) and 18S rRNA served as endogenous control. RNA of mice and HSC samples were isolated using Direct-zol™ RNA MiniPrep (Zymo Research Europe GmbH, Freiburg, Germany) in combination with RNase-Free DNase Set (Qiagen) or NucleoSpin RNA Plus (Macherey-Nagel, Düren, Germany) according to manufacturer's protocols. RNA was reverse transcribed using either cDNA synthesis Kit H Plus (Peqlab, VWR International GmbH, Darmstadt, Germany) or RevertAid RT Reverse Transcription Kit (#K1691, Thermo Scientific). 2μl of cDNA was added in a total volume of 10μl containing Fast-SYBR Green (Applied Biosystems, Foster City, CA, USA) together with specific primers (Supplementary Table 2 and 3) and qPCR was performed on the QuantStudio 6 flex PCR system (Applied Biosystems, Foster City, CA, USA). All results are expressed as 2^−ΔΔCT and represent the x-fold increase of gene expression compared to the control group.

Individual tablets of TRUVADA (tenofovir/emtricitabine; Gilead Sciences) or raltegravir (Merck) were crushed into fine powder and manufactured as 5BXL by TestDiet based as previously published (Halper-Stromberg et al. 2014 ; Cheng et al. 2018 ). HIV-1 RNA was extracted from plasma (Viral RNA Mini Kit, Qiagen). RNA was reverse transcribed and quantitatively detected by real-time PCR using TaqMan® Fast Virus 1-Step PCR kit (ThermoFisher Scientific) and QuantStudio 6 Flex PCR system (Applied Biosystems) (detection limit ≈400 copies/ml) (Li et al. 2014 , 2017 ).

Total RNA was isolated from overnight cultures of UAMS-1 and LAC grown in BFM with and without 0.49-µM telithromycin. RNA was isolated using a Qiagen RNeasy mini kit and reverse-transcribed into cDNA using the iScript cDNA synthesis kit (Bio-Rad Laboratories). RNA concentration and purity were determined using a NanoDrop spectrophotometer (ThermoScientific). Quantitative real-time polymerase chain reaction (PCR) (qRT-PCR) was performed using a QuantStudio 6 Flex PCR system, TaqMan Fast Advanced Master Mix (Applied Biosystems), 25 ng of cDNA, 900 nM of each primer, and 250 nM of the appropriate probe. The full gene expression data set is included as Table 2. The relative fold change in gene expression was calculated using gyrA as a control. Primers and probes used for sarA and gyrA are listed in Table 4.

HIV-1 RNA was purified from the plasma with the QIAamp^® Viral RNA Mini Kit. The RNA was then reverse transcribed and quantitatively detected by real-time PCR using the TaqMan^® Fast Virus 1-Step PCR kit (ThermoFisher Scientific). The primers used for detecting the HIV Gag gene were (5′-GGTGCGAGAGCGTCAGTATTAAG-3′ and 5′-AGCTCCCTGCTTGCCCATA-3′). The probe (FAM-AAAATTCGGTTAAGGCCAGGGGGAAAGAA-QSY7) used for detection was ordered from Applied Biosystems, and the reactions were set up following the manufacturer’s guidelines and were run on the QuantStudio 6 Flex PCR system (Applied Biosystems). The detection limit of the real-time PCR reaction is four copies per reaction. Accordingly, the limit of detection of the assay with 50 µl of blood is 400 copies/ml in humanized mice.

According to the manufacturer’s instructions, the total ribonucleic acid (RNA) in the liver was isolated by RNA-Quick Purification Kit (ES Science, RN001, China) and then synthesized into cDNA by reverse transcriptase (RT) (ES Science, RT001, China). Furthermore, cDNA amplification with appropriate primers to amplification (Table 1) was performed using Super SYBR Green qPCR Master Mix (ES Science, QP002, China) by QuantStudio 6 Flex PCR System (Applied Biosystems, USA). The cycle threshold (Ct) values of indoleamine 2,3-dioxygenase 1 (IDO1), Janus kinase 2 (JAK2), intercellular adhesion molecule 1 (ICAM1), nuclear factor kappa-B inhibitor alpha (NFKBIA), interferon regulatory factor 1 (IRF1), and signal transducer and activator of transcription 1 (STAT1) were obtained and normalized to that of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (all reagents were from TIANYI HUIYUAN, Beijing, China), and the results were analyzed by the 2^−ΔΔCT method.