Light cycler 480 probe master system

Total RNA was isolated using the Quick-RNA Micro prep kit (Zymo) according to the manufacturer’s protocol. Quality control of samples was carried out using a Nanodrop ND-100 Spectrophotometer. Complementary DNA synthesis was performed using the ProtoScript II Reverse Transcription kit (M0368S; New England BioLabs). Quantitative real-time PCR was performed using primers designed with the online tool provided by Roche and the Light-Cycler 480 Probe Master System (Roche). Each reaction was run in duplicates, and relative gene expression levels were normalized to GAPDH. Relative expression was calculated using the ΔΔCt method. Primer sequences used are described in Table S2.

RNA was prepared using RNAeasy Kit (74004, Qiagen) and treated with TURBO™ DNase (AM1907, Ambion) to remove genomic DNA. First-strand cDNA was synthesized using ProtoScript II Reverse Transcriptase (M0368, New England BioLabs). Real-time qPCR primers were designed with the online tool provided by Roche, and quantification was performed using the LightCycler 480 Probe Master System (Roche). Genomic DNA was used as a universal standard to calculate gene copy number per ng of RNA. Relative expression levels were obtained by normalization to Actb mRNA. Following real-time qPCR primers were used: Tas2r143 (forward): 5′-CAG GCA TCT TTT TGA ACT CCA-3′; Tas2r143 (reverse): 5′-TCT TCA GGG CCT TTC TCA GT-3′; Tas2r135 (forward): 5′-CCA TCA TGT CCA CAG GAG AA-3′; Tas2r135 (reverse): 5′-TCA GTA GTC TGA CAT CCA AGA ACT GT-3′; Tas2r126 (forward): 5′-GTG TGT GGG ATT GGT CAA CA-3′; Tas2r126 (reverse): 5′-GCT CCC GGA GTA CTC AAC C-3′; Actb (forward): 5′-AAA TCG TGC GTG ACA TCA AA-3′; Actb (reverse): 5′-TCT CCA GGG AGG AAG AGG AT-3′.

Total RNA was extracted from treated cells with the TRizol reagent (Invitrogen) and cDNA was synthesized according to the manufacturer’s protocol (Roche). Quantitative real-time PCR (qRT-PCR) was performed using a Light Cycler 480 Probe Master System (Roche), and PCR-specific amplification was conducted in the LightCycler® Nano (Roche). The relative expression of genes (PTEN and GAPDH) was calculated with the 2-(ΔΔCt) method. The primers used are listed here (qRT-PCR; PTEN-forward 5′-AAGACAAAGCCAACCGATAC-3′, PTEN-reverse 5′-GAAGTTGAACTGCTAGCCTC-3′; GAPDH-forward 5′-CCTCTATGCCAACACAGTGC-3′, GAPDH-reverse 5′-GTACTCCTGCTTGCTGATCC-3′.

Total RNA was isolated from endothelial cell monolayers using the Quick-RNA Micro prep kit (Zymo) according to the manufacturer’s protocol. Quality control of samples was carried out using a Nanodrop ND-100 Spectrophotometer. Complementary DNA synthesis was performed using the ProtoScript II Reverse Transcription kit (New England BioLabs, M0368S). qPCR was performed using primers designed with the online tool provided by Roche and the Light-Cycler 480 Probe Master System (Roche). Each reaction was run in triplicate, and relative gene expression levels were normalized to GAPDH. Relative expression was calculated using the ΔΔCt method. Primer sequences used were as follows: human Gapdh forward 5′-AGCCACATCGCTCAGACAC-3′, reverse 5′-GCCCAATACGACCAAATCC-3′; human Ccl2(Mcp1) forward 5′-AGTCTCTGCCGCCCTTCT-3′, reverse 5′-GTGACTGGGGCATTGATTG-3′; human Pdgfb forward 5′-CTGGCATGCAAGTGTGAGAC-3′, reverse 5′-CGAATGGTCACCCGAGTTT-3′; bovine Gapdh forward 5′-TCACCAGGGCTGCTTTTAAT-3′, reverse 5′- GAAGGTCAATGAAGGGGTCA-3′; bovine Ccl2(Mcp1) forward 5′-CGTTTGTTTTCATGGAGATTTG-3′, reverse 5′-TCCCAGGGATAGAACTGAGATT-3′; bovine Pdgfb forward 5′-GACTGATGGGGTCGCTGT-3′, reverse 5′-AGGGTTTCAGGCCAGTCC-3′; bovine Sele forward 5′-TGACCAAAGAAGCCACACAAACT-3′, reverse 5′-CACAGTCCTCATCACTTTGCTT-3′.

Total RNA was extracted from prepared cultured cells with TRIzol reagent (Invitrogen) and cDNA was synthesized according to the manufacturer's protocol (Roche). Quantitative real-time PCR (qRT-PCR) was performed using a Light Cycler 480 Probe Master System (Roche), and PCR-specific amplification was conducted using the LightCycler^® Nano (Roche) as described previously^{28 (link)}. The relative expression of CCL26 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was calculated using the 2-(ΔΔCt) method method. The primers and probe kits ofCCL26 and GAPDH were obtained from Applied Biosystems (Nagoya, Japan).