Abi viia7 pcr instrument

RNA was isolated from CD4+ T-cells from patients of different genotypes (PMA and ionomycin stimulated; overnight). RNA isolation was performed using the Allprep DNA/RNA Mini kit (QIAGEN) and cDNA synthesis (for 500 ng RNA) was prepared with Superscript III from Invitrogen. A final concentration of 5 ng was used in quantitative PCR (qPCR), which was performed with the ABI ViiA7 PCR instrument (Applied Biosystems, Paisley, UK) using SYBR Master mix (Applied Biosystems) with evaluation of dissociation curves. mRNA levels of each gene were quantified using the DDCt method and expressed relative to β-actin. For each gene, a TaqMan Gene Expression Assay was used (Life Technologies—according to the manufacturer's instructions); IL23R (Hs00332759_m1), IL12RB2 (Hs00155486_m1), IFNG (Hs00989291_m1), IL10 (Hs00961622_m1) and β-actin (Hs01060665_g1). H19 primers were purchased from Diagenode (Liege, Belgium).

RNA was isolated with TRIzol (Invitrogen, Paisley, UK) and cDNA synthesis (for 500 ng RNA) was prepared with Superscript III from Invitrogen (Paisley, UK). A final concentration of 5 ng/μL was used in qPCR, which was performed with the ABI ViiA7 PCR instrument (Applied Biosystems, Paisley, UK) using SYBR Master mix (Applied Biosystems, Paisley, UK) with evaluation of dissociation curves. mRNA levels of each gene were quantified using the ΔΔCt method and normalised to β-actin. For each gene, PCR melting curves were checked to evaluate the single, specific product.
The specific primers designed were RUNX3 forward: 5′-ACT CAG CAC CAC AAG CCA CT-3′ RUNX3 reverse: 5′-GTC GGA GAA TGG GTT CAG TT-3′ RUNX3 values were normalised to β-actin (Hs_ACTB_1_SG QuantiTect Primer Assay[NM_001101] Qiagen, Manchester, UK).