One step quantifast sybr green rt pcr mix

RNA was extracted from clarified cell culture supernatants (16,000 g x 10 min) using QIAamp Viral RNA^® Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was eluted in 30 μl of RNase-free water and stored at –80 °C until use. The qRT-PCR was carried-out following previously described procedures with minor modifications [13 (link)]. Briefly, reverse transcription and amplification of the S gene were performed using the one-step QuantiFast Sybr Green RT-PCR mix (Qiagen) as follows: 50 °C for 10 min, 95 °C for 5 min; 95 °C for 10 s, 60 °C for 30 s (40 cycles) (primers: RBD-qF1: 5′-CAATGGTTTAACAGGCACAGG-3′ and RBD-qR1: 5′-CTCAAGTGTCTGTGGATCACG-3). Standard curve was obtained by cloning the receptor binding domain of S gene (primers: RBD-F: 5′-GCTGGATCCCCTAATATTACAAACTTGTGCC-3′; RBD-R: 5′-TGCCTCGAGCTCAAGTGTCTGTGGATCAC-3′) into pGEM T-easy vector (Promega, Madison, WI, USA). A standard curve was generated by determination of copy numbers derived from serial dilutions (10³–10⁹ copies) of the plasmid. Each quantification was run in triplicates.

RNA was extracted from clarified cell culture supernatants and infected cells using QIAamp Viral RNA^® Mini Kit (Qiagen, Hilden, Germany) and RNeasy Plus mini kit (Qiagen), respectively, according to the manufacturer’s instructions. The qRT-PCR was carried out following previously described procedures [13 (link)]. Briefly, reverse transcription and amplification of the spike (S) gene were performed using the one-step QuantiFast Sybr Green RT-PCR mix (Qiagen) as follows: 50 °C for 10 min, 95 °C for 5 min; 95 °C for 10 sec, 60 °C for 30 sec (40 cycles) (primers: RBD-qF1: 5′-CAA TGG TTT AAC AGG CAC AGG-3′ and RBD-qR1: 5′-CTC AAG TGT CTG TGG ATC ACG-3). A standard curve was obtained by cloning the receptor-binding domain of the S gene (primers: RBD-F: 5′-GCT GGA TCC CCT AAT ATT ACA AAC TTG TGC C-3′; RBD-R: 5′-TGC CTC GAG CTC AAG TGT CTG TGG ATCAC-3′) into pGEM T-easy vector (Promega, Madison, WI, USA). A standard curve was generated by the determination of copy numbers derived from serial dilutions (10³–10⁹ copies) of the plasmid. Each quantification was run in triplicates.

RNA was extracted from infected cells using RNeasy Plus mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. RNA was eluted in 30 μL of RNase-free water and stored at −80 °C until use. The qRT-PCR was carried out following previously described procedures [35 (link)]. Briefly, reverse transcription and amplification of the S gene were performed using the one-step QuantiFast Sybr Green RT-PCR mix (Qiagen) as follows: 50 °C for 10 min; 95 °C for 5 min; 95 °C for 10 s; 60 °C for 30 s (40 cycles) (primers: RBD-qF1: 5′-CAA TGG TTT AAC AGG CAC AGG-3′ and RBD-qR1: 5′-CTC AAG TGT CTG TGG ATC ACG-3′). A standard curve was obtained by cloning the receptor binding domain of the S gene (primers: RBD-F: 5′-GCT GGA TCC CCT AAT ATT ACA AAC TTG TGC C-3′; RBD-R: 5′-TGC CTC GAG CTC AAG TGT CTG TGG ATCAC-3′) into pGEM T-easy vector (Promega, Madison, WI, USA). A standard curve was generated by the determination of copy numbers derived from serial dilutions (10³–10⁹ copies) of the plasmid. Each quantification was run in triplicates. The 2^−DDCq (Livak) method was used for the comparison of the target qPCR product with the standard curve.