Kapa sybr fast qpcr master mix 5x universal kit

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)}.

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)).

Homogenised lung samples in Trizol were used for RNA isolation using the Trizol-chloroform method as per the manufacturer’s protocol. Isolated lung RNA was quantitated by NanoDrop and 1 µg of total RNA was then reverse-transcribed to cDNA using the iScript cDNA synthesis kit (Biorad; #1708891) (Roche). Diluted cDNAs (1:5) were used for qPCR using KAPA SYBR^® FAST qPCR Master Mix (5X) Universal Kit (KK4600) on Fast 7500 Dx real-time PCR system (Applied Biosystems) and the results were analysed with SDS2.1 software. CDC-approved SARS-CoV-2 N gene primers: 5′-GACCCCAAAATCAGCGAAAT-3′ (Forward), 5′-TCTGGTTACTGCCAGTTGAATCTG-3′ (Reverse) were used for vial load calculation. For absolute quantitation, the known copy number of the virus RNA was used as a standard to generate the calibration curve.

For qPCR from splenocytes or lung samples, RNA isolation and cDNA synthesis was done as described above. Diluted cDNAs (1:5) were then used for qPCR by using KAPA SYBR® FAST qPCR Master Mix (5X) Universal Kit (KK4600) on a Fast 7500 Dx real-time PCR system (Applied Biosystems) and the results were analyzed with SDS2.1 software. Beta-actin gene was used as an endogenous control and was used for normalization through quantitative RT-PCR. The relative gene expression was calculated by -ΔΔCt formula as described previously^{47 (link),52 (link),53 (link)}. The following primer sets were used:
Il-13 – 5’-CTTAAGGAGCTTATTGAGGAG-3’ 3’- CATTGCAATTGGAGATGTTG-5’;; Oas2 – 5’-TTATAAAATACCGGCAGCTC-3’ 3’-ATTACAGGCCTCTTTTTCTG-5’; Oas3 – 5’-CCAAACTTAAGAGCCTGATG-3’ 3’-GCCTCTCCTCCTTTATATCG-5’; RNasel – 5’-ATACTGTAGGTGATCTGCTG-3’ 3’-AAGTATCTCCTTCATTCCCC-5’; CCCTACTCATAAAAATCACCAG-3’ 3’-TTGGAATAGCATTTCCACAG-5’; Ifitm3– 5’-AAGAATCAAGGAAGAATATGAGG-3’ 3’-GATCCCTAGACTTCACGG-5’; β-Actin- 5’-TTAATTTCTGAATGGCCCAG-3’ 3’-GACCAAAGCCTTCATACATC-5’; Il-17 – 5’-ACGTTTCTCAGCAAACTTAC-3’ 3’-CCCCTTTACACCTTCTTTTC-5’; Ifn-γ – 5’-TGAGTATTGCCAAGTTTGAG-3’ 3’-CTTATTGGGACAATCTCTTCC-5’; hACE2–5’-TCCATTGGTCTTCTGTCACCCG-3’; 3’AGACCATCCACCTCCACTTCTC-5’.

Isolated lung was homogenized in 2ml Trizol reagent (Invitrogen) and RNA was isolated by Trizol-Choloform method. Yield of RNA was quantitated by nano-drop and 1 µg of RNA was use to reverse-transcribed to cDNA using the iScript cDNA synthesis kit (BIORAD; #1708891) (Roche). 1:5 diluted cDNAs was used for qPCR by using KAPA SYBR^® FAST qPCR Master Mix (5X) Universal Kit (KK4600) on Fast 7500 Dx real-time PCR system (Applied Biosystems) and the results were analyzed with SDS2.1 software (30 (link), 33 (link)). 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 SARS-CoV-2 N gene: 5′-GACCCCAAAATCAGCGAAAT-3′ (Forward), 5′-TCTGGTTACTGCCAGTTGAATCTG-3′ (Reverse). Hypoxanthine-guanine phosphoribosyl transferase (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 hypoxanthine-guanine phosphoribosyl transferase (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 (36 (link), 37 (link)).