Superscript vilo cdna synthesis system

After stimulation, macrophage cells were lysed in Qiazol (Qiagen) and stored at −80 until RNA isolation. RNA isolation was performed using Direct-zol 96-well isolation column plates (Zymo Research). Approximately 10 ng of RNA was reverse transcribed using Superscript VILO cDNA synthesis system (Invitrogen). 1.25 uL of cDNA was combined with 0.5 uL of a pool of 96 primer sets (Deltagene assays, Fluidigm) at 500nM concentration each, 2.5 uL 2× Taqman Preamp Master Mix (Applied Biosystems), and made up to 5 uL total volume per reaction with water, in a low-profile 96-well PCR plate (Bio-rad). Specific Target Amplification was run on a CFX Connect thermal cycler (Bio-rad). Following pre-amplification, unincorporated primers were digested by the addition of Exonuclease 1 (New England Biolabs). Samples were analyzed by qPCR on the Fluidigm Biomark instrument using 96.96 chips according to manufacturer’s instructions (Fluidigm). Data were exported from Fluidigm Real-time PCR Analysis software version 3.1.3, using Linear (Derivative) Baseline method, a global threshold of 0.01, and a 0.65 quality threshold, parameters which were found to exclude non-specific amplification and reduce plate-plate variation.

All of the clinical samples were subject to HPV typing (13 high-risk and 24 low/intermediate genotypes) by PCR L1 gene for reverse blot hybridization with a linear array (Quest Diagnostics, San Juan Capistrano, CA, USA).
For in-house E6/E7 based typing, total RNA and DNA were isolated from the samples using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and Quick-DNA Universal Kit (Zymo research, Irvine, CA, USA), respectively. Purified RNA was reverse-transcribed into cDNA using SuperScript VILO cDNA Synthesis System (Invitrogen, Carlsbad, CA) with random primer. The PCR reactions for the detection of the genomic DNA and E6 mRNA of HPV contained 2 μl of DNA/cDNA, 200 μM of dNTP, 1.5 mM MgCl₂, I U Taq DNA polymerase, and 200 nM of each primers ^{(8 (link))}. For the multiplex PCR to genotype HPV 6 and 11, one HPV6/11 universal forward primer^{(8 (link))} and two genotype-specific reverse primers (HPV6: 5’-TTA TGA ACC GTG CCT TGG TTA G-3’; HPV11: 5’-CAA CGA CCC TTC CAC TGG TTA-3’) were used. Two sets of primers were used to genotype HPV16^{(8 (link))} and HPV18^{(9 (link))}. The PCR products were resolved in 2% agarose gels and stained with ethidium bromide.