Quantifying HTLV-1 Gene Expression in Blood

Total RNA was extracted from fresh whole blood utilizing TriPure isolation reagent (Roche, Germany). cDNA was synthesized using RT-ROSET Kit (ROJETechnologies, Iran) and SYBR Green-based (TaKaRa, Otsu, Japan) and subsequently, RT-qPCR was performed, according to the manufacturers’ instructions. The following primers were utilized to determine the expression levels of JNK, EVI1, MKP, and PTPRR and to confirm HTLV-1 infected cells in the samples: EVI1 (forward primer (FP): 5′-TCGTCGCCTCATTCTGAACTGGAA-3′, reverse primer (RP): 5′-ACTGCCATTCATTCTCTCCTCCACA-3′) MKP (FP: 5′-AGCCACCATCTGCCTTGCT-3′ , RP: 5′-CCAGCCTCTGCCGAACAGT-3′ ) PTPPR (FP: 5′-CCAGCACTGTCCGAGGCAA-3′ , RP: 5′-GCAAACAGAGGTAGCGGTGGT-3′ ) JNK (FP: 5′-TGCTGTGTGGAATCAAGCACCT-3′ , RP: 5′-TCGGGTGCTCTGTAGTAGCGA-3′ ) HBZ (FP: 5′-ACGTCGCCCGGAGAAAACA-3′ , RP: 5′-CTCCACCTCGCCTTCCAACT-3′) 5’LTR (FP: 5′-GGCTCGCATCTCCCCTTCAC-3′ , RP: GAGCAAGCAGGGTCAGGCAA-3′). The relative two standard curves real-time PCR was performed on the cDNA samples using Q-6000 machine (Qiagen, Germany). The GAPDH gene was utilized to normalize the mRNA expression levels respectively, as well as to control the error between samples [32 (link)]. The output for each group was analyzed using Mann-Whitney U test for statistical difference between gene expression. A p-value of less than 0.05 was considered to be significant.