Itaq supermix

To assess C. trachomatis prevalence in particular, a specific real-time PCR was carried out on all samples to target the omcB gene of C. trachomatis. The reaction was performed in a final volume of 20 µL using iTaq Supermix (BioRad, Reinach, Switzerland), 0.3 µM concentration of each primer, 0.1 µM concentration of probe, and 5 µL sample DNA^{89 (link)}. The program was set at 95 °C for 5 min, followed by 45 cycles of 15 s at 95 °C and 30 s at 60 °C, all carried out in a PikoReal real-time PCR System. All samples were tested in duplicate. Samples with a C_T value of ≤37 were considered as positive. Distilled water (a negative control) and C. trachomatis DNA (a positive control) were included in each experiment.

Total splenocytes were lysed in RLT buffer (#79216, Qiagen) and RNA was isolated using RNeasy Mini Kit (#74104, Qiagen). cDNA was transcribed using Quantitect Reverse transcription kit (#205313, Qiagen) according to manufacturer’s protocol. Ido1 and Ido2 gene expression was measured using iTaq Supermix (#1725124, Bio-rad) using primer the following primers: Gapdh 5’ -CTG CCC AGA ACA TCA TCC CT -3’; 5’ - ACT TGG CAG GTT TCT CCA GG -3’; Ido1 5’ - CGA TGT TCG AAA GGT GCT GC-3’; 5’ - GCA GGA GAA GCT GCG ATT TC-3’; Ido2 5’- CTC AGA CTT CCT CAC TTA ATC G -3’; 5’- GCT CAC GGT AAC TCT -3’. Data were calculated as the ratio to Gapdh expression by relative quantification method (ΔΔct; means ± SD of triplicate determination) and data are presented as normalized transcript expression in the samples relative to normalized transcript expression in control cultures (in which fold change = 1).

RNA was isolated from cells using the GENEzol TriRNA Pure Kit (GeneAid). cDNA synthesis was carried out with the Quanta cDNA Reverse Transcription Kit (QuantaBio). qPCR was performed with the iTaq Supermix (BioRad) on the BioRad iCycler. For BCR-ABL quantification, mononuclear cells derived from peripheral blood of CML patients were isolated on Ficoll-Hypaque gradient and total RNA was isolated. Real-time for BCR-ABL quantification was performed using ipsogen® BCR-ABL1 Mbcr IS-MMR (Qiagen). The comparative Ct method was employed to quantify transcripts, and delta Ct was measured in triplicate. Primers used in this study are provided in Supplementary Table S3.

RNA was isolated from cells using the GENEzol TriRNA Pure Kit (GeneAid). cDNA synthesis was carried out with the Quanta cDNA Reverse Transcription Kit (QuantaBio). Then, qPCR was performed with the iTaq Supermix (BioRad, Hercules, CA, USA) on the Biorad iCycler. The comparative Ct method was employed to quantify transcripts, and delta Ct was measured in triplicate.
Sequences of used primers were the following: Cyclophilin A (Fw: GTCAACCCCACCGTGTTCTT Rv: CTGCTGTCTTTGGGACCTTGT), U2AF65 (Fw: 5′ACCCAGGCTATGGCCTTTG, Rv: 5′GAAGCGGCTGGTAGTCGTG), Jmjd6 total (Fw: 5′GGGTGCGTTAGTGTCAGGAA, Rv: 5′CCTTTCCACGTTATCCGCCA), Jmjd6 Exon 5 Inclusion (Fw: 5′ACCTGGAGGGACCAGCTC, Rv: 5′TCTGAGTCGGAGTCTGACGA), Jmjd6 Exon 5 Exclusion (Fw: 5′CAAGGAAATGGTATAGGATTTTGAA, Rv: 5′TTTGCTGACACAGTCGTCCT).