Total RNA was isolated by using the TRIzol reagent extraction kit (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. Total RNA (1μg) was subsequently reverse-transcribed to cDNA by using the high-capacity cDNA RT kit (Applied Biosystems, Foster City, CA). Diluted cDNA samples were subjected to qPCR using SYBR Selected Master Mix kit (Applied Biosystems, Foster City, CA) and specific primers for individual genes (ABI Prism TM 7900HT Sequence Detection System, Applied Biosystems, Foster City, CA). Relative mRNA expression levels were calculated using the ΔΔCt method and normalized to a housekeeping gene RPLP0 (60S acidic ribosomal protein P0). Sequences of all primers used for amplification reaction assays are summarized in Table 1 .
Abi prism tm 7900ht sequence detection system
Manufactured by Thermo Fisher Scientific
The ABI Prism TM 7900HT Sequence Detection System is a real-time PCR instrument designed for high-throughput gene expression analysis and SNP genotyping. The system utilizes fluorescence detection technology to monitor the amplification of DNA sequences in real-time.
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2 protocols using abi prism tm 7900ht sequence detection system
1
Gene Expression Analysis by qPCR
2
Validating CNVs via Quantitative PCR
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The CNVs were validated by quantitative PCR (qPCR). Quantitative PCR validation was performed using the ABI PrismTM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). Three pairs of primers were selected from the start, middle and end of each CNV, separately. The sample was analysed in triplicate in a 10 μl reaction mixture (200 nM each primer, Maxima® SYBR Green/ROX qPCR Master Mix (2X) from Fermentas, and 5 or 10 ng of genomic DNA). The values were evaluated using System 7900 Software SDS2.3 (Applied Biosystems, CA). Further data analysis was performed using the qBase method. Reference genes, chosen from COBL, GUSB, and SNCA, were included based on the minimal coefficient of variation, and the data were then normalized by setting a normal control to a value of 1.
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