Quantitative RT-PCR for SARS-CoV-2 Gene Expression

Isolated lung was homogenized in 2 ml of Trizol reagent (Invitrogen) and RNA was isolated by the Trizol-Chloroform method. RNA yield was quantitated by nano-drop and 1 µg of RNA was used to reverse-transcribe cDNA using the iScript cDNA synthesis kit (BIORAD; #1708891) (Roche). cDNAs (1:5 dilution) were used for qPCR by using the KAPA SYBR^® FAST qPCR Master Mix (5X) Universal Kit (KK4600) on the Fast 7500 Dx real-time PCR system (Applied Biosystems), and the results were analyzed with SDS2.1 software (23 (link), 37 ). Briefly, 200 ng of RNA was used as a template for reverse transcription-polymerase chain reaction (RT-PCR). The CDC-approved commercial kit was used for of the SARS-CoV-2 N gene: 5′-GACCCCAAAATCAGCGAAAT-3′ (Forward), 5′-TCTGGTTACTGCCAGTTGAATCTG-3′ (Reverse). The hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene was used as an endogenous control for normalization through quantitative RT-PCR. The relative expression of each gene was expressed as fold change and was calculated by subtracting the cycling threshold (Ct) value of the HGPRT-endogenous control gene from the Ct value of the target gene (ΔCT). Fold change was then calculated according to the formula POWER(2,-ΔCT)*10,000 (74 (link), 75 (link)).