Taqman universal pcr master mixture

Based on the CNV results of the NanoString nCounter assay, quantitative PCR (qPCR) analysis was performed on the three most frequent genes using commercially available, predesigned TaqMan Copy Number Assays (Fig. 1). To allow a comparison between both approaches, we selected qPCR probes focused on the immediate region of the NanoString's probes. The reaction mixture contained 2 mL genomic DNA template, 10 mL of Taqman universal PCR master mixture (Applied Biosystems, Foster City, CA, USA), and 0.2 mM of each primer.
To accurately detect copy number gain (CNG), we analyzed three different regions of the CCND1, KRAS, and MDM2 genes (Supplementary Table 1). CNG was determined using the following qPCR sequence: 2 min at 50 ˚C, denaturation at 95 ˚C for 10 min, followed by 40 cycles of 95 ˚C for 15 s and 60 ˚C for 1 min using relative quantification in a 7900 HT fast real-time PCR system in quadruplicate. A RNaseP assay kit (Applied Biosystems) was used as a control. After amplification, the resulting threshold cycle values for the copy number and reference assay were imported into the CopyCaller Software (Applied Biosystems) for post-PCR data analysis as previously described. CNG and the number of copies were determined based on concordance of the results with one or more of the three regions of each probe.

RNA from three biological cell culture controls was isolated with the RNeasy Kit (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. One microgram of cellular RNA was reverse transcribed into cDNA using SuperScript II reverse transcriptase and random hexamer primers (Invitrogen/Life Technologies, USA). The PCR reaction was carried out in a mixture that contained appropriate sense- and anti-sense primers and a TaqMan MGB probe in Taq-Man Universal PCR Master Mixture (Applied Biosystems, Foster City, CA, USA) (see Supplementary File). Beta-2-microglobulin was used as housekeeping gene. qRT-PCR amplification and data analysis were performed using the Lightcycler 96 (Roche Applied Science, Penzberg, Germany). Each sample was assayed in duplicate. The ΔΔCq method was used to determine relative gene expression levels.

Total RNA was isolated using TRIzol (Invitrogen, CA, USA), and 0.5 µg of total RNA was reverse transcribed into cDNA and PCR amplified using a one-step RT-PCR kit with SYBR Green (Applied Biosystems, Grand Island, NT, USA). Real-time quantitative PCR was performed using a real-time PCR system (7500, Applied Biosystems). Table 1 shows the primer sequences and probe sequences. cDNA was amplified using a TaqMan® Universal PCR master mixture containing DNA polymerase (Applied Biosystems, Foster, CA, USA) according to the manufacturer’s instructions. The concentration of the target gene was determined using the comparative Ct (threshold cycle number at the cross-point between the amplification plot and the threshold) method (Table 1).