SARS-CoV-2 N gene quantification from lung samples

RNA isolated from lung or other organ samples homogenized in Trizol (Invitrogen) was used for viral load estimation. Quantitation of isolated RNA was done and 1 µg of total RNA was then reverse-transcribed to cDNA using the iScript cDNA synthesis kit (Biorad; #1708891) (Roche). 1:5 diluted cDNAs were then used for qPCR by using KAPA SYBR® FAST qPCR Master Mix (5X) Universal Kit (KK4600) along with the on a Fast 7500 Dx real-time PCR system (Applied Biosystems) and the results were analyzed with SDS2.1 software. Briefly, cDNA was used as a template for the CDC-approved commercial reagent for SARS-CoV-2 N gene: 5′-GACCCCAAAATCAGCGAAAT-3′ (Forward), 5′-TCTGGTTACTGCCAGTTGAATCTG-3′ (Reverse). Beta-actin gene was used as an endogenous control and was used for normalization through quantitative RT-PCR. The region of N gene of SARS-CoV-2 starting from 28287 – 29230 was cloned into pGEM®-T-Easy vector (Promega). This clone was linearized using SacII enzyme and in vitro transcribed using the SP6 RNA polymerase (Promega). The transcript was purified and used as a template for generating a standard curve to estimate the copy number of SARS-CoV-2 N RNA as previously described^{50 ,51 (link)}.