Macaque infection was confirmed by SIVgag nested PCR on PBMC as described [49 (link)]. Plasma samples were obtained from EDTA-treated whole blood and used for the determination of plasma VL by SIVgag qRT-PCR [50 (link)] (quantitative Molecular Diagnostics Core, AIDS and Cancer Virus Program Frederick National Laboratory). DNA and RNA were extracted from snap frozen tissues using DNeasy/RNeasy blood and tissue kits (Qiagen) following the manufacturer’s instructions. Tissue viral DNA loads were quantified using the standard curve method and normalized by albumin copy numbers by Gag-qPCR as described in [59 (link)]. For tissue RNA loads, 1μg of total RNA was retrotranscribed to DNA using the VILO Kit (Thermo Fisher) quantified by Gag-qPCR [59 (link)].
Dneasy rneasy blood and tissue kits
Manufactured by Qiagen
The DNeasy/RNeasy blood and tissue kits are a series of products designed for the efficient extraction and purification of DNA or RNA from a variety of biological samples, including blood, tissues, and cultured cells. These kits utilize a silica-based membrane technology to selectively bind and concentrate the desired nucleic acids, while effectively removing contaminants and inhibitors. The extracted DNA or RNA can then be used for various downstream applications, such as PCR, sequencing, or further analysis.
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4 protocols using dneasy rneasy blood and tissue kits
1
Quantifying SIV Infection in Macaques
2
SIV Detection and Quantification in Macaques
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Macaque infection was confirmed by SIVgag nested PCR on PBMC as described [49 (link)]. Plasma samples were obtained from EDTA-treated whole blood and used for the determination of plasma VL by SIVgag qRT-PCR [50 (link)] (quantitative Molecular Diagnostics Core, AIDS and Cancer Virus Program Frederick National Laboratory). Tissue viral DNA and RNA loads were measured, respectively, by qPCR and qRT-PCR with standard curve method and normalized on Albumin copy number (for cell-associated viral DNA) and total RNA quantity. DNA and RNA were extracted from snap frozen tissues using DNeasy/RNeasy blood and tissue kits (Qiagen) following the manufacturer’s instructions. Primers: SIVgag FW (5’-GGTTGCACCCCCTATGACAT-3’), SIVgag RV (5’-TGCATAGCCGCTTGATGGT-3’); macaque Albumin FW (5’-ATTTTCAGCTTCGCGTCTTTTG-3’) and RV (5’-TTCTCGCTTACTGGCGTTTTCT-3’). For the DNA loads the Mastermix was ABsolute Blue Q-PCR SYBRGreen low-ROX (Thermo Fisher Scientific, Waltham, MA), while for RNA loads we used the One-step RT-qPCR Kit (KAPA Biosystems, Wilmington, MA) [14 (link)]. The PCR was run on ViiA-7 Real-Time PCR System (Thermo Fisher).
3
Quantifying SIV Viral Loads in Plasma and Tissues
4
Quantifying SIV Viral Loads in Plasma and Tissues
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Blood was collected in EDTA tubes, and plasma separated by density gradient centrifugation was used for the determination of plasma VL by SIVgag qRT-PCR at NIRC or at Leidos (Quantitative Molecular Diagnostics Core, AIDS and Cancer Virus Program Frederick National Laboratory). Tissue VL from snap-frozen PBMC pellets, colorectal biopsies and LN FNA were quantified as described in ref. 78 (link). Briefly, tissue viral DNA and RNA loads were measured, respectively, by qPCR and qRT-PCR with standard curve method and normalized to Albumin copy number (for cell-associated viral DNA) and total RNA quantity. DNA and RNA were extracted from snap-frozen tissues using DNeasy/RNeasy blood and tissue kits (Qiagen) following the manufacturer’s instructions. Primers: SIVgag FW (5’-GGTTGCACCCCCTATGACAT-3’), SIVgag RV (5’-TGCATAGCCGCTTGATGGT-3’), SIVProbe (5’-6-FAM-AATCAGATGTTAAATTGTGTGGGA-3’); macaque Albumin FW (5’-ATTTTCAGCTTCGCGTCTTTTG-3’), RV (5’-TTCTCGCTTACTGGCGTTTTCT-3’), Probe: (5’-6-FAM-CCTGTTCTTTAGCTGTCCGTG-3’). SIV-IPDA was performed on freshly stored PBMC before cycle 1 and at the end of cycle 4 of galunisertib by Accelevir, Baltimore, MD.
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