Random hexanucleotide primer

Total RNA extraction and purification from TERA1 and NT2/D1 cell lines, testicular cancer and normal testis samples were performed as described above. First strand cDNA synthesis was carried out with the random hexanucleotide primer (Promega, USA) and MintReverse Transcriptase (Evrogen, Russia) according to the manufacturers' protocols. For RT-qPCR, reactions were performed using qPCRmix-HS SYBR system (Evrogen, Russia) on Lightcycler 480 (Roche, USA) in accordance with the manufacturers' instructions. DNA fragments were amplified for 40 cycles of 95°C for 20 s, 60°C for 20 s, 72°C for 20 s. Relative level of mRNA was quantified with 18S rRNA serving as the reference. Technical triplicates were used to ensure reproducibility. For RT-PCR, reactions were performed using Encyclo Polymerase Mix (Evrogen, Russia) on Biorad DNAEngine PTC (Biorad, USA). DNA fragments were amplified for 35 cycles of 95°C for 20 s, 60°C for 20 s, 72°C for 3 min. Primer pairs used in amplification are listed in Table S4.

RNA extraction and purification from the normal testis sample were performed according to Sambrook et al.[64] . First strand cDNA synthesis was carried out with the random hexanucleotide primer (Promega, USA) and MintReverse Transcriptase (Evrogen, Russia) according to the manufacturers' protocols. cDNA for each PIWIL2 control isoform was PCR amplified with primers containing EcoRI and NotI restriction enzymes recognition sites (Table S6) to facilitate further cloning of the amplification product into pCI vector (Promega, USA). Transfections were performed using Lipofectamine 2000 (Invitrogen, USA) as recommended by the manufacturer. Cells were lyzed 48 hours after the transfection and probed by Western blot analysis. Biological duplicates were used to ensure reproducibility.

Reverse transcription of total RNA was performed at 37 °C using the Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA) and random hexanucleotide primers (Promega, Madison, WI). Real-time quantitative PCR analyses were performed on a Light Cycler 480 SYBR Green I master and a Light Cycler 480 apparatus (both from Roche Diagnostics, Indianapolis, IN). The PCR product integrity was verified by melting curve analysis. Quantification data were normalized to the amplification data for the reference gene encoding ribosomal protein S9 (RPS9). The sequences of the primers for IL-15, GZMB, PRF1, IFNγ, CD206, F4/80, TGFβ, and RPS9 are presented  (Additional file 1: Table S1).