7900 ht real time pcr

RNA from treated or untreated cells was extracted using Trizol (Thermo Fisher Scientific, MA USA) following manufacturer’s instructions. 200 ng of total RNA from each sample were retro-transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystem, Thermo Fisher Scientific, MA USA). qPCR reactions were performed by means of a 7900 HT Real Time PCR (Applied Biosystem) using gene specific primers for the following selected genes:
BAX: Forward 5′-TTTGCTTCAGGGTTTCATCCA-3′: Reverse 5′- CTCCATGTTACTGTCCAGTTCGT-3′; BCL-2: Forward 5′-GTTCCCTTTCCTTCCATCC-3′; Reverse 5′-TAGCCAGTCCAGAGGTGAG-3′; FAS: Forward 5′-CCCTCCTACCTCTGGTTCTTACG-3′; Reverse 5’TCAGTCACTTGGGCATTAACACTTT-3′; FASL: Forward 5′-CCTGAAAAAAAGGAGCTGAGGAA-3′; Reverse 5′-GGCATGGACCTTGAGTTGGA-3′; GAPDH: Forward 5′-CAAGGCTGTGGGCAAGGT-3′; Reverse 5′-GGAAGGCCATGCCAGTGA-3:
Primers were designed at exon-exon junctions using Primer express 2.0 (Applied Biosystems). Target expression level was performed as previously described [19 (link)] using GAPDH as housekeeping gene. All the experiments were performed in triplicate. Data are expressed as the mean ± SD.

RNAs were isolated from the cortex and striatum from one half of the brain of R6/2 mice, at different disease stage, and of age-matched WT control mice using Trizol, (Life Technologies). Tissue homogenization was performed using Tissue lyser (Qiagen) following manufacturer instructions. RNA quality was assessed using Experion system (BioRad). For qPCR assays, from each sample 200 ng of total RNA were retro-transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystem). q-PCR was performed by means of a 7900 HT Real Time PCR (Applied Biosystem). Gene specific primers for the selected genes (Table 1) were designed at exon-exon junctions using Primer express 2.0 (Applied Biosystems). mRNA expression levels of TJ proteins were normalized over Cyclophilin A, used as internal control. Normalized values in HD tissues were then expressed as fold of change of normalized values in WT tissues. The entire procedure for q-PCR analysis, including primer design, reactions, amplicon specificity, and determination of gene target expression levels, was performed as previously described16 (link).

RNA extraction and q-PCR were essentially performed as previously described [19 (link)]. Brefly, Total RNA was isolated from each sample with Trizol (Thermo Fisher Scientific, Waltham, MA USA), as indicated by manufacturer. For each sample to analyse, cDNA was than obtained starting from 200 ng of total RNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystem, Thermo Fisher Scientific, Waltham, MA USA). The described selected genes using gene specific primers
BAX: Forward 5′-TTTGCTTCAGGGTTTCATCCA-3′: Reverse 5′- CTCCATGTTACTGTCCAGTTCGT-3′; BCL- 2: Forward 5′-GTTCCCTTTCCTTCCATCC-3′; Reverse 5′-TAGCCAGTCCAGAGGTGAG-3′; p53: Forward 5’-TCTGTCCCTTCCCAGAAAACC-3’; Reverse 5’-CAAGAAGCCCAGACGGAAAC-3′; GAPDH: Forward 5′-CAAGGCTGTGGGCAAGGT-3′; Reverse 5′-GGAA GGCCATGCCAGTGA-3’.
All primers were selected using a specific software (Primer express 2.0, Applied Biosystems, Foster city, CA, USA) and all of them specifically covered the exon-exon junctions. The analysis of gene expression was done as described in [20 (link)] and GAPDH gene was used as internal control. qPCRs were done using the 7900 HT Real Time PCR (Applied Biosystem) and for each experimental condition a triplicate was performed. Data obtained are expressed as the mean ± SD.