Rnx plus kit

Total RNA was isolated from denuded Oocytes (n = 20) using the RNX‐Plus kit (cinnagen, RN7713C). The cDNA was synthesized using Add Script cDNA Synthesis Kit (ADD BIO, Korea). The exclusive primers of each marker were designed using Allele ID 7 software (Table 1) and synthesized by Takapoo Zist Tehran Company.
The primer solution was made at a concentration of 100 pmol/μl. The GAPDH gene is used as a control gene to determine the relative expression of the genes studied. The best temperature of PCR‐Gradient was determined for each gene by Allele ID 7 software (Table 2). The cDNA was synthesized using a kit (Real Q plus Master Mix Green‐low Rox A 3244‐2). The cDNA (2 μl) was added to SYBR green master mix (10 μl), forward (5 μM) and reversed (5 μM) primers.

The mRNA expression levels of widely established apoptotic and antiapoptotic related genes, caspase-3 and Bcl-2, were performed using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). After subculture and treatment, total cellular RNA was isolated from the untreated and treated cells using an RNX PLUS Kit (Cinnagen, Iran) according to the manufacturer's protocol. Then the quality and quantity of isolated RNA were evaluated by a NANODROP 2000c spectrophotometer (Thermo Scientific, USA). Subsequently, the RNA was reverse transcribed into cDNA and used as the template for PCR amplification using a reverse transcriptase kit (Thermo Scientific, USA). Quantitative RT-PCR (qRT-PCR) was performed by the Corbett Rotor-Gene 6000 system (Corbett Life Science, Australia). PCR was carried out in a final volume of 20 μL reaction system containing 0.2 μM of each primer (Table 1), 10 μL of SYBR green reagent (RR820L Takara Bio, Japan), 1 μL of cDNA template, and 8.6 μL of nuclease-free water.
The PCR cycling was carried out by initial denaturation step at 95°C for 3 min followed by 45 cycles at 95°C for 10 seconds, 58°C for 30 seconds, and 72°C for 20 seconds. Relative mRNA expression was measured by the 2^−(ΔΔCT) method, using β-actin and GAPDH as reference genes [21 (link)].

The expression of Plzf and Sycp3 genes were assessed
by real-time PCR. For extraction of total RNA from
samples, RNX-Plus™ KIT (Cinna Gen, Iran) was used,
then RNA was treated with DNase I (Fermentase,
USA) to remove the genomic contamination. The RNA
concentrations were measured by a biophotometer
(Eppendorf, USA). cDNA was synthesized from 1000 ng
RNA using a cDNA kit (Fermentase, Germany) (21 (link)).
Primers for Plzf and Sycp3 genes were designed using the
NCBI website and were synthesized by Cinna Gen (Iran,
Table 1). The PCR reactions were done using Master Mix
and SYBR Green (Fluka, Switzerland) in a StepOne™
thermal cycler (Applied Biosystems, USA). Melting curve
analyses were used for confirmation of the quality of the
PCR reactions. A standard curve was used to determine
the efficiency of each gene (logarithmic dilution of cDNA
from the samples). In addition, this process was repeated
in triplicates for all the target and reference (ß-actin)
genes. The target genes were normalized to the reference
gene.

RNAs were extracted by means of RNX™ Plus Kit (Cinnagen) from the treated seedlings according to manufacturer's instructions. Next to DNaseI treatment of RNA samples, 1 μg of RNAs, using RevertAid First Strand cDNA Synthesis Kit (Thermo SCIENTIFIC, # K1691), was reverse transcribed to corresponding cDNAs, which were later used as templates for semi-quantitative RT–PCR.