One step reverse transcriptase pcr kit

Using total RNA extracted from monocyte enriched PBMCs (1x10⁶ cells, Ambion, Life Technologies, Carlsbad, CA, USA), reverse transcriptase-PCR was performed on RNA (50 ng) with a one-step reverse transcriptase-PCR kit (Qiagen, Hilden, Germany) using gene-specific primers for IL-12p40, ARG1, CD206, Peroxisome proliferator activated receptor gamma (PPARG), Vitamin D receptor (VDR), 25-Hydroxyvitamin D₃ 1-alpha-hydroxylase (CYP27B1), LL-37 (cathelicidin) and β-actin (S1 Table). Primers were designed using NCBI gene bank reference sequences of human genes and their specificity for humans were confirmed by Basic Local Alignment Test (BLAST) in NCBI. The amplification cycle comprised 35 cycles of denaturing (94°C for 30 seconds), annealing for 30 seconds (varying temperature for each primer set; S1 Table), extension (72°C for 60 seconds), and a final extension at 72°C (10 minutes). Products were resolved on agarose gels (2%) containing ethidium bromide (0.5 mg/mL), observed and analyzed in G-BOX gel doc [Syngene, Cambridge, UK] using Gene Tools (Version 4.01.04) software. The values were normalized to β-actin, which was considered as 100% for each individual.

Skin biopsies were stored in RNALater at −80 °C and total RNA isolated as per the manufacturer’s instructions (Ambion, Life Technologies, USA). Reverse transcriptase-PCR was performed on RNA (50 ng) with the one-step reverse transcriptase-PCR kit (Qiagen, Hilden, Germany) using gene-specific primers (10 µM) for β-actin (F, 5′-CCCAAGGCCAACCGCGAGAAAGAT-3′; R, 5′-GTCCCGGCCAGCCAGGTCCAG-3′), and PD-1 (F, 5′-ATGTAGCCGCCCCACACAGA-3′; R, 5′-CATCCATCTTTTTCAGCCAT-3′). After incubation at 50 °C for 30 min. and PCR activation (95 °C, 15 min.), the amplification comprised 35 cycles of denaturing (94 °C, 30 seconds), annealing (30 seconds), extension (72 °C, 60 seconds), and a final extension (72 °C, 10 min.) in a thermocycler (Applied Biosystems, California, USA). Products were resolved on agarose gels (2%) containing ethidium bromide (0.5 μg/ml) and analyzed (G-BOX gel doc, Syngene, Cambridge, UK) using Gene Tools (Version 4.01.04) software, values being normalized to their respective β-actin.

RVA in the stool samples was detected using ProSpecT™ enzyme immunoassay (EIA) kit (Oxoid, Basingstoke, UK) following the manufacturer’s instructions. RVA positive samples were amplified in the VP4 and VP7 segments using One-step Reverse Transcriptase PCR Kit (Qiagen, Valencia, CA, USA) using previously published primers [45 (link),46 (link)]. Successful amplification of the target regions was confirmed by the presence of expected bands (VP4: 660 bp and VP7: 881 bp) following gel electrophoresis of the PCR products. Products from successful PCRs were purified using GFX DNA purification kit (GFX-Amersham, Amersham, UK) and sequenced bi-directionally (both in forward and reverse directions) using Big Dye Terminator 3.1 (Applied Biosystems, Foster City, CA, USA) chemistry. The primers used during PCR amplification were used for sequencing on an ABI Prism 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).