Superscript 3 one step rt pcr system kit

RNA was recovered from the cultured macrophages with TRIzol Reagent (Invitrogen) according to the manufacturer’s specifications. For reverse-transcription PCR (RT-PCR), 1 μg of total RNA was retro-transcribed and amplified with a couple of gen-specific primers at 50 pM using the SuperScript III One-Step RT-PCR System kit (Invitrogen). Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) (housekeeping gene), Arg1, nitric oxide synthase 2 inducible (Nos2), and Chil3 genes were amplified using the primers designed previously [28–30 (link)]. The amplicons were electrophoresed on 1% agarose gel and stained with Ethidium Bromide. Gels were visualized and images captured in a Kodak Electrophoresis Documentation and Analysis System (EDAS) 120. The relative transcription of genes was determined by densitometry quantitation of the bands observed for each group in the gels using ImageJ software, values are presented as arbitrary units.

Amplification and Sanger sequencing of a larger fragment of the RdRp and VP1 gene were carried out on NoV positive samples as previously described^{10 (link),29} . Briefly, one-step RT-PCR was carried out using “SuperScript III One-Step RT-PCR System kit” with Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA) following thermal profile was applied: retro-transcription at 50 °C for 60 min, initial denaturation at 94 °C for 2 min followed by 40 cycles with Taq activation at 94 °C for 15 sec, annealing at 55 °C for 30 sec, extension at 68 °C for 90 sec and final elongation at 68 °C for 5 min.
The sequences obtained were compared with those available using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and nt sequences were deposited into GenBank under accession numbers: MN605609-MN605633.