Steponeplus rt qpcr machine

cDNA was prepared by first strand synthesis using the TURBO DNA-free Kit (REF AM1907; Invitrogen) and Superscript III (REF 5675; Invitrogen). First, 2.5 µg of total RNA was treated with DNase I and incubated at 37°C for 30 min. After inactivation with DNase Inactivation Reagent, 2 μl of DNase-treated RNA was combined with 4.5 μl of a master mix containing random hexamers Roche and dNTPs. Samples were incubated at 65°C for 5 min, then placed on ice. While on ice, 3.5 μl of RT master mix (1X first-strand buffer, DTT, RNaseOUT, SSIII [REF 56575; Invitrogen]) was added to each tube, then samples were incubated at 25°C for 5 min, 50°C for 1 h, and 70°C for 15 min. For RT-qPCR, 5.2 μl Absolute Blue SYBR Green ROX (REF AB-4162; Thermo Fisher Scientific) and 4.8 μl cDNA (diluted 1:20 prior to use) was added to each well of qPCR plates (REF 4346906; Life Technologies). Samples were run on a StepOnePlus RT-qPCR machine (Applied Biosystems). Average C_T values of triplicate reactions were used to calculate expression changes. RT-qPCR data was normalized to ACT1 for mitotic experiments and PFY1 for meiotic experiments (primers listed in Table S3).

The RNA was extracted from brain and cell lines with TriZOL reagent (15596018, ThermoFisher Scientific) per ThermoFisher protocol, precipitated from 100% isopropanol and washed in 70% ethanol. RNA was quantified and purity assessed (260 nm: 280 nm value between 1.8 and 2.2) by UV-Vis spectroscopy (Beckman Coulter DU 640 Spectrophotometer). cDNA was synthesized using 2.5 μM Oligo dT 20 primer (18418020, ThermoFisher Scientific) and Superscript III Reverse Transcriptase (18080044, ThermoFisher Scientfic) per ThermoFisher published protocol. RT-qPCR was performed with iQ SYBR Green Supermix (Bio-Rad) using a StepOnePlus RT-qPCR machine (Applied Biosystems) with gene specific primers (Supplementary Table S1). mRNA levels were normalized to GAPDH. Relative levels of mRNAs IP’ed were then normalized to overall mRNA levels in lysate. Ct values obtained were verified to be within the linear concentration range obtained for primers (see MIQE in Supplementary Files). A sample lacking reverse transcriptase (RT-) and a non-template control (NTC) were run for each sample/ primer set and verified to have non-detectable fluorescence. Melt curves were analyzed to verify a single peak at the appropriate melt temperature. Experiments were performed at least three times and a two-tailed T-test was performed to determine significance.