Taqman mrna assay

High-Capacity cDNA Reverse Transcription Kit (Life Technologies) and TaqMan mRNA assays (Life Technologies) were used to perform reverse transcription of mRNAs coding for the following proteins: ITGA1 (assay ID: Mm01306375_m1), ITGA2 (assay ID: Mm00434371_m1), KLRG1 (assay ID: Mm00516879_m1), GZMC (assay ID: Mm01313651_m1), GZMN (assay ID: Mm00461851_m1), KLRD1 (assay ID: Mm00495182_m1), KLRB1F (assay ID: Mm04211785_m1), and CLEC2I (assay ID: Mm00777071_g1). PCR reactions were carried out with the TaqMan^® gene expression master mix (Life Technologies) according to the instructions given by the manufacturer on a 7900HT real-time PCR System, as previously described [44 (link)]. Raw Ct values were calculated using the SDS software v.2.4 with GAPDH (assay ID: Mm99999915_g1) for normalization. Fold change of expression was calculated with the comparative Ct method (2-ΔΔCt) [53 (link)]. Data sets were analyzed for statistical significance using two-tailed unpaired heteroskedastic Student’s t-test (* = p<0.05).

Total RNA from human and mouse islets were prepared as described [20 (link)]. CFTR mRNA expression was measured by RT-qPCR using primers and probes from Taqman mRNA assays (Life Technologies, California, USA). Human CFTR: Hs00357011_m1 expression was normalized against human HPRT1: 4333768 F, while mouse CFTR: Mm00445197_m1 expression was normalized against mouse HPRT1: Mm00446968. RT-qPCR runs were performed for individual batches of human (N = 5) and mouse (N = 5) islets in triplicate wells of 384-well plate on a 7900 HT RT-PCR system (Applied Biosystems, California, USA).

Using High Capacity cDNA Reverse Transcription Kit (Life Technologies) and TaqMan mRNA assays (Life Technologies) we performed reverse transcription of mRNAs coding for the following proteins: RHD (assay ID: Mm00456910_m1), ERMAP (Mm_00469273_m1), ADD2 (Mm00478923_m1), ANK1 (Mm00482889_m1), PLK1 (Mm00440924_g1), CCNA2 (Mm00438063_m1), LIFR (Mm00442942_m1), and CD163 (Mm_00474091_m1). PCR reactions were performed with the TaqMan® gene expression master mix (Life Technologies) according manufacturer's protocol on a 7900HT real-time PCR System, as described previously (Al-Quraishy et al., 2014 (link)). All samples were run in duplicates and raw ct values were calculated using the SDS software v.2.4 and GAPDH was used for normalization. Fold change of expression was calculated with the comparative Ct method (2^−ΔΔct) (Livak and Schmittgen, 2002 (link)). Data sets were analyzed for statistical significance using two-tailed unpaired heteroskedastic Student's t-test (^*p < 0.01).

Quantitative real-time PCR was performed under experimental conditions identical to those described recently [28 (link)], using High Capacity cDNA Reverse Transcription Kit (Life Technologies) and TaqMan mRNA assays (Life Technologies) for reverse transcription of mRNAs encoding the following proteins: ERMAP (assay ID: Mm00469273_m1), CLDN13 (Mm00491038_s1), CD163 (Mm00474091_m1), GZMB (Mm00442837_m1), KLRB1A (Mm00726548_s1), KLRC3 (Mm00650941_m1), KLRD1 (Mm00495182_m1), NCR1 (Mm01337324_g1), KLRG1 (Mm00516879_m1), and GAPDH (Mm99999915_g1). Fold change of expression was calculated using the comparative Ct method (2^−ΔΔct) [29 (link)] and data sets were analysed for statistical significance using a two-tailed unpaired heteroscedastic Student’s t test (*p < 0.01).

For real-time qPCR, the Applied Biosystems TaqMan^® Fast Advanced Master Mix was used (Cat. No. 444557, Thermo Fisher Scientific, Waltham, MA, USA), alongside specific TaqMan^® mRNA Assays (Cat. No. 4331182, Thermo Fisher Scientific, Waltham, MA, USA) for each gene: IL-1A (Hs00174092_m1), IL-1RN (Hs00893626_m1), IL-6 (Hs00174131_m1), IL-10 (Hs00961622_m1), TNF-A (Hs00174128_m1), IFN-G (Hs00989291_m1), SIRT1 (Hs01009006_m1), SIRT3 (Hs00953477_m1), GSS (Hs00609286_m1), SOD2 (Hs00167309_m1), ICAM1 (Hs00164932_m1), and B2M (Hs00187842_m1). Each sample was assayed in duplicate. The PCR protocol began with a cycle at 95 °C for ten minutes, followed by 40 cycles of 95 °C for 15 s and 60 °C for one minute. Fluorescence signals were collected at the end of each cycle to determine cycle threshold (Ct) values. The fold changes in mRNA expression were calculated using the relative quantification (RQ) method, where ΔCt sample = Ct_target − Ct_HK, ΔΔCt = ΔCt sample − average ΔCt control group, and RQ = 2^−ΔΔCt [23 (link)]. Fold-change values above one suggest up-regulated gene expression, while values below one indicate down-regulation.