Total rna mini kit

Total RNA was extracted using Total RNA Mini kit (Frogga Bio Inc, Canada) as described [58 (link)]. RNA concentrations were measured by NanoDrop 2000c UV-VIS Spectrophotometer (Thermo Scientific, USA) under 260 nm. Equal amount of RNA was reverse-transcribed using SuperScript Ш First-Strand Synthesis System (Invitrogen, USA) to obtain cDNA. The cDNA was diluted (1:5 dilutions) and used as templates for real-time PCR using a SYBR Green PCR Kit (QIAGEN). The PCR was performed for 40 cycles: denaturation at 95°C for 5 sec, annealing at 59°C for 30 sec, and elongation at 72°C for 30 sec (ABI PRISM 7700 Sequence Detection System, USA). The mRNA levels of Foxo3a, Bim, Fas and Fas L were analyzed using U6 as an internal control. All tests were performed in triplicate.

Total RNA was extracted using Total RNA Mini kit (Frogga Bio Inc, Canada) as described [16 (link), 43 (link)]. The RNA concentration was measured by NanoDrop 2000c UV-VIS Spectrophotometer under 260 nm. Equal amount of RNA was used for reverse-transcription to obtain cDNA for real-time PCR amplification. The cDNA was diluted as template and performed in a real-time thermal cycler using a SYBR Green PCR Kit as follows: 40 cycles of denaturation at 95°C for 5 sec, annealing at 59°C for 30 sec, followed by an elongation step at 72°C for 30 sec. The Foxo3 was analyzed using U6 as an internal control for product normalization. All tests were performed in triplicate.

Total RNA from testes of e14.5, e17.5, and 1 dpp animals was extracted using the Total RNA Mini Kit (FroggaBio, Concord, ON, Canada) according to the manufacturer’s protocol. Total RNA was reverse-transcribed using 100 ng of RNA and the SuperScriptVilo™ cDNA synthesis kit (Thermo Fisher scientific, Waltham, Ma, U.S.). Real-time PCR reactions were run on a CFX96 Touch instrument (Bio-Rad, Hercules, CA, USA), using Supergreen Advanced qPCR MasterMix (Wisent, St-Bruno, Qc, Canada). Each PCR reaction consisted of 7.5 μL of Power SYBR Green PCR Master Mix, 2.3 μL of water, 4 μL of cDNA sample and 0.6 μL (400 nmol) of gene-specific primers. PCR reactions run without cDNA (water blank) served as negative controls. A common thermal cycling program (3 min at 95 °C, 40 cycles of 15 s at 95 °C, 30 s at 60 °C and 30 s at 72°C) was used to amplify each transcript. To quantify relative gene expression, the Ct of genes of interest was compared with that of Rpl19, according to the ratio R = [E^{Ct Rpl19}/E^{Ct target}] where E is the amplification efficiency for each primer pair. Rpl19 Ct values did not change significantly between tissues, and Rpl19 was therefore deemed suitable as an internal reference gene. The specific primer sequences used are listed in Supplemental Table S2.