Quantitative qpcr sybr green reagents

The cDNA was diluted at 1:50 in RNAse-free water. Real-time quantitative PCR (RT-qPCR) was performed using a Bio-Rad CFX-96 detection system and quantitative qPCR SYBR Green reagents. A total of 10 μl of 2X Power SYBR^® Green Master Mix (Applied Biosystems, Thermo Fisher Scientific) was mixed with 1 μl of both forward and reverse primers (10 μM) and 5 μl of diluted cDNA (final volume: 20 μL). The result was incubated at 95°C for 10 min to activate the polymerase. The primer sequences are provided in Table 2. All the PCRs were performed in duplicate under the following conditions: 40 cycles of 95°C for 15 s and 60°C for 45 s. The relative levels of the target genes were normalized using the mean expression of 36B4, Hprt1, and Ywhaz as a reference. Relative gene expression was quantified using the 2^–ΔΔCT method.

Total RNA was prepared using RNeasy mini kit (Qiagen) and treated with DNase I, Amp Grade (Invitrogen, Carlsbad, CA, USA) prior to cDNA synthesis. RNA integrity was electrophoretically verified by ethidium bromide staining. RNA (1 μg) was reverse transcribed with 100 U of Superscript II plus RNase H⁻ Reverse Transcriptase (Invitrogen) using 100 μM random hexamer primers (Amersham Biosciences, Piscataway, NJ, USA), according to the manufacturer's instructions. Real-time quantitative PCR was carried out on a Bio-Rad CFX-96 detection system with quantitative qPCR SYBR Green reagents (Bio-Rad, Hercules, CA, USA) and with a primer concentration of 0.5 μM. For a list of primer sequences, see Supplementary Table 1. PCR conditions were standardized to 39 cycles of: 95°C for 08 s, 59°C for 05 s and 72°C for 10 s.

Total RNA was prepared using a RNeasy mini kit (Qiagen) and treated with DNase I, Amp Grade (InVitrogen, Carlsbad, CA, USA) prior to cDNA synthesis. RNA integrity was electrophoretically verified by ethidium bromide staining. RNA (0.5 µg) was reverse transcribed with 100 U of Superscript II plus RNase H- Reverse Transcriptase (InVitrogen) using 100 µM random hexamer primers (Amersham Biosciences, Piscataway, NJ, USA), according to the manufacturer's instructions. Real-time quantitative PCR was carried out on a Bio-Rad CFX-96 detection system with quantitative Q-PCR SYBR Green reagents (Bio-Rad, Hercules, CA, USA) and with a primer concentration of 0.5 µM. PCR conditions were standardized to 40 cycles of 95°C for 10 s and 59°C for 30 s with the primers for specific mouse mRNA sequences (For a list of primer sequences, see Table S1). To control for RNA quality and cDNA synthesis, β-actin or Nono mRNA was also amplified. The abundance of each RNA normalized to the HPRT1 or Nono signal depending on the tissue are expressed as the mean ± SEM of at least 6 samples.