Lc fast start dna master sybr green 1 mix

Total RNA from the 10⁵ hMSCs (passages 3–6) was extracted using the TRIzol reagent (Invitrogen). cDNA synthesis was performed using a SuperScript III system (Invitrogen). The qPCR was carried out on a LightCycler 480 system (Roche Diagnostics) using an LC-FastStart DNA Master SYBR Green I mix (Roche). In addition, 5 µL of each cDNA was rapidly mixed with 1 µL of forward and reverse primers and 13 µL of LC-FastStart DNA Master SYBR Green I mix. The amplification profile was as follows: enzyme activation at 95°C for 10 min; and annealing at 95°C for 10 s, 60°C for 5 s, and 72°C for 15 s. The specificity of the PCR products was determined by a melting curve analysis. The forward and reverse primers of the human genes were designed using LightCycler Probe Design Software 2 (Roche), and sequences are shown in table 1. Results are expressed relative to the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

RNA was extracted from frozen samples of either tumoral or matched non-tumoral parts of HCC patient biopsies using the RNeasy mini kit (Qiagen, Hilden, Germany); 0.25 μg of total RNA was reverse-transcribed with the QuantiTect Reverse Transcription kit (Qiagen), and cDNA was quantified using LC Fast start DNA Master SYBR Green I Mix (Roche, Basel, Switzerland). Primers for RT-qPCR were designed using LightCycler probe design 2 software (Roche), targeting the pan-genotype small (i.e., 300 bp) conserved region in the 5′UTR of HCV viral sequence: forward: 5′- CAGGAGATGGGCGGAAAC -3′, reverse: 5′- GCCGCAATGGATATTTCATTCTCA-3′. Results were normalized to SRSF4 housekeeping gene expression; forward: 5′-CGGAGTCCTAGCAGGCATA-3′, reverse: 5′-TTCCTGCCCTTCCTCTTGT-3′. GOLT1B expression was quantified using qPCR primers GOLT1B-fw (5′-CGGCTTCATTTCTCCCGACT-3′) and GOLT1B-rv (5′-TCCAATTTTCTGCGTGTCCG-3′) using the SYBR green method using a CFX96 Touch Thermal Cycler (Bio-Rad, Hercules, CA, USA). PCR products were validated by melting curve analysis and Sanger sequencing.