Hipure total rna plus mini kit

For the determination of cell-associated HIV-1 RNA, total RNA was extracted from peripheral blood mononuclear cells (PBMCs) using a Magen HiPure Total RNA Plus Mini Kit (Guangzhou, China) according to the manufacturer protocol, followed by the quantification of the contained HIV-1 RNA using a SUPBIO HIV-1 usRNA Quantitative PCR kit (Guangzhou, China). Plasma HIV-1 RNA was determined using the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test (version 2.0) following standard procedures. A third-party laboratory performed the two determinations.

The total cellular DNA and RNA were extracted from isolated PBMC samples by QIAamp DNA Mini Kit (Qiagen, Valencia, California) and HiPure Total RNA Plus Mini Kit (Magen, Guangzhou, China), respectively, and then amplified and quantified with the HIV DNA and cell-associated RNA (ca-RNA) quantitative detection kits (SUPBIO, Guangzhou, China) on the Roche LightCycler 480 (LC480) real-time PCR platform. The quantification of HIV caDNA and caRNA was multiplied by the percentage of CD4⁺ T cells in PBMC and the quantification of DNA/RNA copies among 1× 10⁶ cells.

The mRNA expression of NLRP3, IL-1β, and MMP-13 in retrieved corneas was detected by performing real-time RT-PCR on day 5. The corneas were placed in a 1.5 ml centrifuge tube, and total RNA was extracted according to the HiPure Total RNA Plus Mini Kit (Magen). One microgram of total RNA was reverse transcribed using the PrimeScript™ RT reagent kit with gDNA Eraser (Takara Biotechnology Co.). The used primer pair sequences were synthesized by TSINGKE Biological Technology (Beijing, China) and are listed in Table 1. qPCR was performed using SYBR® Premix Ex Taq™ II (Takara Biotechnology Co.) with a QuantStudio5 quantitative PCR instrument (Applied Biosystems). Transcript quantification was performed in triplicate for each sample. Results were reported relative to the control group after normalized by GAPDH using the △△CT method.

Total RNA was extracted from leukemia cells using the HiPure Total RNA Plus Mini Kit (R4121‐02, Magen). Using total RNA as a template, reverse transcription was conducted and cDNA was synthesized with TransScript All‐in‐one first‐strand cDNA Synthesis SuperMix for qPCR (AT341‐01, TransGen). According to the instructions of ChamQ Universal SYBR qPCR Master Mix (Q311‐03, Vazyme), qPCR was performed using a Bio‐RAD CFX96 fluorescence quantitative PCR instrument and the relative expression of the interested genes was calculated by the 2ˆ(ΔΔCt) method. Primers were designed according to the PrimerBank website (https://pga.mgh.harvard.edu/primerbank/). qPCR primers are shown in Table S4 of the Supporting Information.

RNA was extracted with a HiPure Total RNA Plus Mini Kit (Magen). Library construction and RNA sequencing were constructed by Novogene with an Illumina HiSeq2000 (150 bp, paired-end). The sequencing data were qualified by fastqc (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and the differentially expressed genes (DEG) called using the RNA Cocktail framework [25 (link)].

Total cellular DNA and RNA were extracted from PBMCs using the Qiagen QIAsymphony DNA Mini Kit (Qiagen, Valencia, CA, USA) and HiPure Total RNA Plus Mini Kit (Magen, Guangzhou, China), respectively. A fluorescence-based real-time SUPBIO HIV Quantitative Detection Kit (SUPBIO, Guangzhou, China) was used to amplify and quantify the HIV DNA and RNA. The DNA and RNA copy numbers were normalized to 1 × 10⁶ PBMCs.

RNA was extracted with the HiPure Total RNA Plus Mini Kit (Magen). cDNA was synthesized using a HiScript III All-in-one RT SuperMix Perfect for qPCR kit (Vazyme) according to the manufacturer’s protocol. QPCR assays were performed in triplicates with the ChamQ Universal SYBR qPCR Master Mix (Vazyme). The relative quantitation (RQ) of gene expression was normalized to GAPDH. The qRT-PCR primers are listed in Supplementary Table 5.