Procedure
Total RNA was extracted from OHSCs by Trizol (Invitrogen), following the instructions provided by the manufacturer, and was quantified by spectrophotometric analysis. Total RNA (1 lg) from each sample was transcribed into cDNA using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, Eugene, OR) according to the manufacturer’s instructions. Real-time PCR was performed on the RT products with SensiMix SYBR Kit (Bioline), or (for arginase-1 mRNA expression) with SensiMix II probe Kit (Bioline), following the manufacturer’s instructions, using an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA). Primer sequences (from MWG Operon) and accession numbers are listed in Table 1 and in Cacci et al., 2008. Annealing temperature was 60C for all the primer pairs listed. All samples were run in triplicate, and each well of PCR contained 20 ll as a final volume of reaction, including 2 ll of cDNA corresponding to 20 ng of total RNA, 0.75 lM of each primer, and 10 ll of PCR master mix. Thermal cycling conditions were as follows: one cycle at 95C for 10 min; 40 cycles 95C for 15 sec and 60C for 1 min. Expression levels of genes of interest were compared between control unstimulated and stimulated cultures using the relative quantification (DDCt) study of Applied Biosystems 7500 System SDS Software. Hypoxanthine guanine phosphoribosyl transferase (HPRT) was used as internal control gene and amplification specificity was checked using a melting curve, following the manufacturer’s instructions.
Stats
- Recommendations n/a n/a positive of 0 vote(s)
- Views 702
- Comments 0