Beacon designer 8

The qRT-PCR was conducted using the iQ5 real-time PCR system (Bio-Rad). The gene-specific primers (Table 1) were designed using the Beacon Designer 8.0 software (Premier Biosoft International). Each primer pair (Tm 60°C) was designed to amplify an approximately 200-bp fragment. For each sample, 1 μL cDNA, 1 μL each primer, 2 μL double-distilled water, and 5 μL 2x SYBR Premix ExTaq II (Takara) were used in a total volume of 10 μL. The two-step RT-PCR was completed using the manufacturer’s recommended program, but the annealing temperature was changed to 60°C. Samples were heated at 95°C for 10 s, cooled to 65°C for 15 s, and finally heated to 95°C at a rate of 0.1°C s^–1 for melting curve analyses. The specific transcript accumulation was analyzed using the 2^–ΔΔCT method (Livak and Schmittgen, 2001 (link)). Peach 18S ribosomal RNA was used to normalize data. The amplification, melt curve and melt park of 18s ribosomal gene in all samples can be seen in Supplementary Figure S1. Each sample was analyzed in triplicate.

A quantitative analysis of gene expression was carried out using the Applied Biosystems 7500 Real-Time PCR System (Life Technologies, USA) with the qRT-PCR master mix with EVA Green stain and ROX passive reference dye (Sintol, Russia). The following protocol was used: denaturation at 95°C for 5 min, followed by 40 cycles at 95°C for 15 sec and at 60°C for 1 min. All experiments were run in triplicate. The expression levels of target mRNAs were normalized to the expression of the reference gene hypoxanthine-guanine phosphoribosyltransferase (Hprt). The relative levels of target gene expression were calculated using the comparative 2-^ΔΔCt method (ABI Relative Quantification Study software, Applied Biosystems, USA). Specific primers were designed based on GenBank and Ensemble data concerning the annotated sequences of the target genes using the Beacon Designer 8.0 software (Premier Biosoft, Palo Alto, CA, USA) (Supplementary Table 6). Due to high homology of mouse Mage genes, the primers were designed to detect the expression of five of eight genes the Mage-a subfamily and all genes of the Mage-b,Mage-d, Mage-e, Mage-l and Mage-h subfamilies The designed Mage primers were pre-screened using cDNAs synthesized from total RNAs from adult mouse testes (Supplementary Figure 4 and Supplementary Table 7).

For qRT-PCR, 5 μg of RNase-free DNase I-treated RNA was processed with M-MLV reverse transcriptase (Takara Bio Inc., Japan) according to the manufacturer’s instructions. Relevant PCR primers directed against a selection of differentially transcribed sequences identified by iTRAQ (Additional file 1) were designed using Beacon Designer 8.0 software (Premier Biosoft International, Palo Alto, CA, USA). A fragment of the gene encoding 18S rRNA was used as a reference. PCR was performed with a Bio-Rad iQ5 instrument (Bio-Rad, Hercules, CA, USA), using SYBR Green to detect transcript abundance. Each 25-μL reaction contained 0.5 μM of each primer and approximately 0.5 U of enzymes, cDNA and SYBR Green. Negative control reactions contained no cDNA. Five-fold dilutions of the cDNA templates were tested under conditions identical to those used for the samples being tested. The PCR regime included an initial denaturing step (95 °C/10 s), followed by 40 cycles at 95 °C/5 s, 60 °C/10 s, and 72 °C/15 s and a final stage at 55 °C to 95 °C to determine dissociation curves of the amplified products. All reactions were replicated at least three times. The data were analysed using Bio-Rad iQ5 Optical System Software v2.1. The relative transcript level of each gene was calculated using the 2^-△△CT method, and the data were normalized based on the 18S rRNA CT values [43 (link)].