Itaq universal sybr green pcr master mix

Quantitative real time PCR assays were performed with samples that were collected independently of samples used for RNA-Seq analysis. Three biological replicates were used for each genotype/treatment. RNAs were treated with RQ1 RNase-Free DNase (Promega) and reverse-transcribed using Improm II reverse transcriptase (Promega) and oligo(dT)15 primer (Promega) according to the manufacturer’s recommendations. The qRT-PCR was performed by using the QuantStudio^TM 6 Flex Real-Time PCR System (Applied Biosystems) and iTaq Universal SYBR Green PCR master mix (Bio-Rad). Relative transcript levels were calculated by the comparative delta-Ct method and normalized to the ACT2 (At3g18780) gene transcript level. The primers used in this study were designed by Primer Express Software for Real-time PCR, Version 3.0 (Applied Biosystems) and the primer sequences are included in Supplementary Table S6.

A total of 3000 Mastl^+/+ and Mastl^−/− PGCs sorted by FACS were used to prepare total RNA with the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Oligo (dT)-primed cDNA was synthesized using the iScript cDNA synthesis kit (BioRad, Hercules, CA, USA). Real time PCR for Mastl was performed using the primers 5′-
GGA GTA TCT TAT TGG TGG AGA-3′ and 5′-
AGC ATA TTG TCC GGT TTC AA-3′ on a CFX-Connect Real-Time system (Bio-Rad) using iTaq Universal SYBR Green PCR master mix (Bio-Rad). The reaction was performed in triplicate. Gapdh was used as the internal control. The PCR products were run on a 3% agarose gel with ethidium bromide.

Total RNA was extracted using the Spectrum Plant Total RNA Kit (Sigma-Aldrich) and quantified spectrophotometrically at 260 nm using the NanoDrop 2000 (Thermo Fisher Scientific). One µg of RNA was treated with RQ1 RNase-Free DNase (Promega) and reverse-transcribed using Improm II reverse transcriptase (Promega) and oligo(dT)₁₅ primer (Promega) according to the manufacturer’s recommendations. The qRT-PCR was performed with iTaq Universal SYBR Green PCR master mix (Bio-Rad) on a QuantStudio^TM 6 Flex Real-Time PCR System (Applied Biosystems). Relative transcript levels were calculated using the comparative delta-Ct method and normalized to the transcript levels of ACTIN2 (At3g18780). The primers used in this study are listed in Supplementary Table 3.

The plants were grown in half MS medium with 0.7% agar. Five- and ten-day-old seedlings were collected in 2 ml tubes and frozen in liquid nitrogen. After homogenization, total RNA was extracted using the Trizol reagent and quantified by using the NanoDrop 2000 (Thermo Fisher Scientific). For mRNA expression analysis, 1 μg total RNA was used for reverse transcription with oligo dT using the Superscript III RT kit (Invitrogen) according to the manufacturer’s instructions. The qRT-PCR was performed with iTaq Universal SYBR Green PCR master mix (Bio-Rad) on a CFX96 real-time PCR detection system (Bio-Rad). Each 20 μl reaction mixture was composed of 10 μl SYBR Green (BioRad), 1 μl of 10 μM primers (forward and Reverse), 6 μl deionized H₂O, and 2 μl cDNA. The qPCR cycling condition was set at initial denaturation of 30 s at 95 °C followed by 40 cycles of denaturation at 95 °C for 10 s and annealing at 60 °C for 45 s. After the completion of amplification, melt curve was produced to determine the primer specificity at 65 °C for 5 s, followed by heating up to 95 °C with 0.5 °C increment. Relative transcript levels were calculated using the comparative delta-Ct method and normalized to the transcript levels of ACTIN2 (At3g18780). The primers used in this study are listed in Supplementary Table 2.