Abi 7900ht fast rt pcr instrument

For qRT-PCR from brain tissue, dissections and RNA extraction were performed as described in Seigneur and Südhof (2017) (link). VeriQuest Probe One-Step qRT-PCR Master Mix with ROX (Affymetrix) and an ABI 7900HT Fast RT-PCR instrument (Applied Biosystems) were used. Samples were run in technical triplicates with Gapdh and Actb as loading controls. Data were normalized to the arithmetic mean of Gapdh and Actb, and mean 2^−ΔCt values are plotted.

Real-time qRT-PCR assays of independent patient cohorts of NSCLC tumor samples were used to further select biomarkers to form a multi-gene assay from prognostic genes identified from microarray data in our previous studies [[18] (link), [19] , [20] (link), [21] (link)]. The identified prognostic genes were initially validated with multiple independent NSCLC microarray data publically available [[18] (link), [19] , [20] (link), [21] (link)]. Based on the validation results, 160 prognostic genes and three housekeeping genes were included in the qRT-PCR experiments. The three housekeeping genes were 18S, UBC, and POLR2A due to their confirmed constant mRNA expressions across samples [18 (link)].
We analyzed 337 tumor samples with good RNA quality using TaqMan microfluidic low-density array (LDA) plates on an ABI 7900HT Fast RT-PCR instrument (Applied Biosystems). Total RNA samples were analyzed on an Agilent 2100 Bioanalyzer RNA 6000 Nano LabChip. The report was generated by the SDS2.3 software (Applied Biosystems). In the report, the number of cycles required to reach threshold fluorescence (Ct) and ∆C_T for each sample relative to the control gene defines the expression pattern for a gene. The gene expression data were further analyzed using the 2^−ΔΔC_T method [22 (link)].