QIAamp viral RNA mini assay was selected after comparisons with other commercially available extraction assays for its reliability and efficacy.
RNA was extracted from 500 µl HIV-1 positive plasma specimens using QIAamp Viral RNA Mini Assay (Qiagen, Valencia, CA).
Briefly, RNA was extracted from 140 µl of serum concentrated from 500 µl, using QIAamp Viral RNA Mini Assay (Qiagen, Valencia, CA).
Direct submission of the RNA (minus DNase treatment) from both the matrices to Conv-RT-qPCR analysis (RT minus assay) gave C T values in the 10-15 cycle range.
When the same RNA samples were subjected to serial DNase I treatment, the C T values changed to 35-38 (DNase R1), 41-42 (DNase R2) and no detection (DNase R3), when analyzed under similar assay conditions, i.e. RT minus assay using Conv-RT-qPCR.
The SNPs used to develop the mini-assays were selected from the 44 K SNP database and additional re-sequencing data based on their distribution across the genome and their ability to detect polymorphism in the specific parental accessions used to construct the CSSLs.
For gel electrophoresis insoluble particles were removed by centrifugation for 2 min at 14,000 g and the supernatant was quantified using BCA mini-assay.
After the incubation wells were washed and the bound 125I-fibrin was quantified by measuring the whole well in a mini-assay type 6 20 gamma counter Mini-instruments Ltd , Burnham-on-Crouch, UK).
To investigate if IVS2-1G>T is responsible for exon-2 skipping; a mini gene assay was performed using the four expression plasmids containing the CYP2C93 gene fragment from exon 1 to exon 3. Two plasmids (pcDNA3.1-mfCYP2C93_IVS2-1G and pcDNA3.1-mfCYP2C93_IVS2-1T) contained the CYP2C93 gene fragment of cynomolgus monkey (mfF1), with IVS2-1G and IVS2-1T, respectively.
The mini BCA assay (ThermoScientific, Ottawa, ON, Canada) was used to determine the protein concentration of each fraction, prior to equal loading in 15% SDS-polyacrylamide electrophoresis gel and Western blotting.
For the mini genome assay, transduced cells were transfected, in a 24-well format, with pCAGGS constructs for IAV WSN/33 PB2, PA2, PA (100 ng each), and NP or empty pCAGGS (200ng), as well as the RNA polymerase II-driven Renilla luciferase reporter pRLTK (40 ng), and the IAV-specific RNA polymerase I-driven firefly luciferase reporter (pPolI-luc, 60 ng).
