Abi prism 7700 sds

RNA was extracted from whole cochleae freshly isolated from Cx30^+/+ and Cx30^−/− mice at P5 using an RNeasy kit (Cat. No. 74104, Qiagen, Milan, Italy). cDNA was obtained by reverse transcription of mRNA with Oligo(dT)12–18 (Cat. No. 18418012, Thermo Fisher Scientific, Milan, Italy) and OmniScript Reverse Transcriptase (Cat. No. 205111, Qiagen) for 1 h at 37°C. qPCR was performed on cDNA to amplify Cx26, Cx30, and p53 and normalized to GAPDH expression (Pfaffl, 2001 (link)). Amplification was carried out using SYBR Green (Cat. No. 4367659, Applied Biosystems) on the ABI 7700 sequence detection system equipped with the ABI Prism 7700 SDS software using the following amplification cycles:

Total RNAs were isolated from unstressed and stressed seedlings. The preparation of cDNA was accomplished using SuperScript^® VILO™ cDNA Synthesis Kit (Invitrogen). An aliquot of 2.5 μg RNA was used as initial sample for cDNA preparation, and diluted cDNA was used for RT-PCR. RT-PCR was performed with the ABI PRISM 7700 SDS (Applied Biosystems, USA) using SYBR Green PCR Master Mix (Brilliant III Ultra-Fast SYBR R, Green Q PCR master mix, Agilent technologies) in a final volume of 20 μl, including cDNA template (10–20 ng) and appropriate primer pairs (final concentration 900 nM) (Supplementary Table 1). The parameters were as follows: 10 min at 95 °C, and 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Three technical and biological repeats were performed for each of the candidate genes. Ubiquitin, actin and EF1α were used as the internal standards.