Faststart sybr green master rox

RNA was isolated from snap frozen tissues using the RNeasy mini kit (QIAGEN). RNA concentration and quality were determined with NanoDrop (Thermo Fisher Scientific). Reverse transcription was performed using the QuantiTect Reverse Transcription kit (QIAGEN), according to the manufacturer’s instructions. qRT-PCR was performed on a ViiA 7 Real-Time PCR System (Life Technology). Amplification was performed with Fast Start SYBR Green Master (ROX; Roche) with 0.5 µM of each forward and reverse primers for the target of interest or appropriate normalization genes (Table S2) and cDNA as template, using the following conditions: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. After each run, a melting curve analysis was performed, using the following conditions: 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. For all steps, a ramp rate of 1.6°C/s was used. Raw Ct values were used to calculate relative expression levels of target genes, after normalization to three internal control genes (Eif2a, Utpc6, and Gapdh) according to geNorm (Vandesompele et al., 2002 (link)). Eif2a and Utpc6 primer sequences were originally reported in (Kosir et al., 2010 (link)); Gapdh primer sequences were obtained from the Genomics Platform–University of Geneva.

Total RNA (1 μg) was reversely transcribed into cDNA using Quantitect Reverse Transcription Kit (Qiagen) according to the manufacturer’s protocol. For mRNA expression analysis real-time PCR was performed (reactions in duplicates) using Fast Start SYBR Green Master Rox (Roche). Real-time PCR was performed on an ABI PRISM 7900 HT and VIIA7 Fast Real-Time PCR System (AB). Data were generated and analyzed using SDS 2.4 and RQ manager 1.2 software. mRNA expression levels were normalized to the housekeeping genes Hprt for human samples, and Gapdh for murine samples.

DNA content and RNA-expression level were determined by qRT-PCR. Total DNA was extracted by the classic phenol/chloroform-based method. Total RNA extraction and reverse transcription were performed with the Maxwell 16 LEV Plant RNA Kit (Promega) and ReverTra Ace (Toyobo), respectively. qRT-qPCR was performed using FastStart SYBR Green Master (ROX, Roche Diagnostics) and the Mx3000P system following the manufacturer’s instructions. The quantitative estimations were calculated with MxPro software using the ΔΔCt (cycle threshold) method (Stratagene, Agilent Technologies), using a nuclear gene MAA7 (SI Appendix, Figs. S4 and S5) or GBLP (SI Appendix, Fig. S7) sequence as an internal control. The list of primers used in this study is shown in SI Appendix, Table S3.