Quantitect human tslp primer assay

Total RNA was extracted from liver and skin samples with the RNeasy Mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol, and reverse transcribed to cDNA using the iScript Select cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA). Five microliters of cDNA were used in a nested PCR for HCV RNA detection together with the AmpliTaq Gold PCR master mix (Applied Biosystems, Foster City, CA, USA) and the following primer sets targeting the 5′-untranslated region: sense 5′-GGGGGCGACACTCCACCA-3′ (position 15 to 32) and anti-sense 5′-TCGCGACCCAACACTACTC-3′ (position 256 to 274) for the first round of amplification; and the inner primers sense 5′-GAGTGTCGTGCAGCCTCCAG-3′ (position 98 to 117) and anti-sense 5′-CTCGGCTAGCAGTCTCGCGG-3′ (position 239 to 258) for the second round.
An additional PCR for TSLP mRNA was carried out using 5 μl cDNA, the AmpliTaq Gold PCR master mix (Applied Biosystems), and the QuantiTect human TSLP primer assay (Qiagen). This primer set targets exons 1/2/3 and amplifies a region of 94 base pairs in the two transcripts (2,629 base pairs and 3,834 base pairs, respectively).

TSLP mRNA was measured in patient samples by absolute real-time RT-PCR quantification. The reaction was performed using the QuantiTect human TSLP primer assay (Qiagen) and FastStart DNA Master SYBR Green I (Roche Diagnostics, Mannheim, Germany) on a LightCycler 1.5 instrument (Roche Diagnostics), according to the manufacturer’s protocol. The cycling conditions were 95°C for 15 minutes, 45 cycles of 94°C for 15 seconds, 55°C for 20 seconds, and 72°C for 20 seconds. The final concentration of TSLP mRNA was expressed as copies of mRNA per nanogram of total RNA.
Real-time RT-PCR for HCV RNA quantitation was performed using the RNA Master HybProbe kit (Roche Diagnostics) and the following primers and probes: 5′-AGCGTCTAGCCATGGCGT (sense), 5′-CAAGCACCCTATCAGGCAGT (antisense), 5′-LC640-CCCGGGAGAGCCATAGTGGTCTG--PH (5′LCRed640) and 5′-GCAGCCTCCAGGACCCCCC--FL (3′HCV-G-Fitch) (TIB MOLBIOL, Berlin, Germany). To generate a standard curve, serial dilutions of the AcroMetrix HCV Mid Control (AcroMetrix, Benicia, CA, USA) were prepared. The HCV RNA concentration was calculated as International Units per nanogram of total RNA; the sensitivity was 20 IU. Each sample and standard curve point was run in duplicate.