Revatra ace

Total RNA was prepared using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA), according to the manufacturer's instructions. Subsequently, 500 ng of total RNA was used to generate cDNA using the RevaTra Ace reverse‐transcription reagents (TOYOBO, Osaka, Japan), according to the manufacturer’s instructions. qPCR was performed using commercially available gene‐specific PrimeTime qPCR probes (listed below; purchased from INTEGRATED DNA TECHNOLOGIES, Coralville, CA, USA) and 2 × Thunderbird Probe qPCR mix (TOYOBO), following the manufacturer's instructions. Gene‐expression levels were normalized to those of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). The following PrimeTime qPCR probes were used (Hs, Human probes): C‐X‐C motif chemokine ligand 1 (CXCL1), Hs.PT.58.39039397; GAPDH, Hs.PT.39a.22214836; actin alpha 2, smooth muscle (ACTA2), Hs.PT.56a.2542642; Interleukin 6 (IL6), Hs.PT.58.40226675; leukemia inhibitory factor (LIF), Hs.PT.58.27705899; connective tissue growth factor (CTGF), Hs.PT.58.14485164.g; tropomyosin‐1 (TPM‐1), Hs.PT.58.39747432. For the co‐culture samples, RFP‐labeled Capan‐1 and GFP‐labeled AD‐MSC cells were isolated from the co‐culture using fluorescence‐activated cell sorting (FACS)‐Aria III (BD Bioscience, Franklin Lakes, NJ, USA), according to the manufacturer's instructions, and then analyzed.

Total RNA was isolated using Sepasol^® (Nacalai Tesque, Kyoto, Japan) and isolated total RNA was used to synthesize complementary DNA using RevaTra Ace^® (TOYOBO, Shiga, Japan), as previously described [27 (link)]. Quantitative real-time PCR analysis based on the intercalation of SYBR Green (Nippon Genetics Co., Ltd., Tokyo, Japan) was performed as previously described [18 (link)]. The following primer sequences were used: Gapdh-F 5′-ATGTGTCCGTCGTGGATCTGA-3′, Gapdh-R 5′-CCTTCTCCATGGTGGT-3′ (NM_001289726, 145 bp), Klkb1/PSA-F 5′-TGGTCGCCAATGGGTACTG-3′, Klkb1/PSA-R 5′-ATATACGCCACACATCTGGATAGG-3′ (NM_008455, 71 bp), TMPRSS2-F 5′-AAGTCCTCAGGAGCACTGTGCA-3′, TMPRSS2-R 5′-CAGAACCTCCAAAGCAAGACAGC-3′ (NM_015775, 116 bp), AR-F 5′-TTGCAAGAGAGCTGCATCAGTT-3′, AR-R 5′ACTGTGTGTGGAAATAGATGGGC-3′ (NM_013476, 153 bp), Probasin-F 5′-GGTCATCATCCTCCTGCTCA-3′, Probasin-R 5′-AGGCCCGTCAATCTTCTTTTT-3′ (NM_017471, 79 bp). The housekeeping gene Gapdh served as an internal control and was used to normalize the differences in the input RNA.

Gene expression levels were quantified by real-time PCR method. Total RNA was extracted from skeletal muscle tissues, liver and HUVECs using RNeasy Mini Kit (Qiagen). RNA which had an OD260/280 ratio of 1.8 or greater was used for reverse transcription reaction. cDNA was produced from 0.5 μg total RNA using a Revatra Ace (Toyobo) [24 (link)]. PCR was performed with a Bio-Rad real-time PCR detection system using THUNDERBIRD SYBR qPCR Mix as a double-standard DNA-specific dye. Primers were 5'-GCTCCAAGCAGATGCAGCA-3' and 5'-CCGGATGTGAGGCAGCAG-3' for mouse 36B4, 5'-GCTGCTGGAGGACGGTTACA-3' and 5'-CACAGGTCCCCAGGATGTTG-3' for mouse FGF21, 5'-GCCCAGCAACATTATCCAGT-3' and 5'-GGTCAGACTTCCTGCTACGC-3' for mouse LPL, 5'-CGGAGTCCGGGCAGGT-3' and 5'-GCTGGGTAGAGAATGGATGAACA-3' for mouse TNF-α, 5'-GCTACCAAACTGGATATAATCAGGA-3' and 5'-CCAGGTAGCTATGGTACTCCAGAA-3' for mouse IL6, 5'-GCCTGTGTTTTCCTCCTTGC-3' and 5'-CTGCCTAATGTCCCCTTGA-3' for mouse IL1β, 5'-CCACTCACCTGCTGCTACTCAT-3' and 5'-TGGTGATCCTCTTGTAGCTCTCC-3' for mouse MCP1 and 5’-AGGTTGGATGGCAGGC-3’ for mouse adiponectin. All results were normalized to 36B4.

Polymorphism analysis for DLA-DRB1 was performed using RNA samples from the 403 dogs as templates by using DLA-DRB1 specific primer sets (DRB1-F and DRB1-R) (Supplementary Table S1D) [31 (link)]. The cDNA samples were synthesized with the oligo-dT primer using the RevaTra Ace reverse transcriptase reaction (TOYOBO, Osaka, Japan) after DNase I treatment using 1 μg of RNA (Invitrogen/Life Technologies/Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturer’s protocol. The polymorphism analysis was also performed using DNA samples from the 426 dogs as templates by using DLA-DRB1 specific primer sets (DRB1-g-F and DRB1-g-R) (Supplementary Table S1E) [32 (link)]. The composition of the PCR solution consisted of 20 ng of cDNA or genomic DNA, 0.4 units of KOD FX DNA polymerase, 10 uL of 2× PCR buffer, 2 mM of dNTP, and 0.4 uM of each primer in 20 uL. The cycling parameter was as follows: an initial denaturation with 94 °C/1 min followed by 33 cycles of 98 °C/10 s, 60 °C/30 s, and 68 °C/45 s.

To exclude any contamination of the feeder cells, ES cells and iPS cells were passaged twice on gelatin-coated dishes. Subsequently, the total RNA was isolated from the cells using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The extracted RNA was treated with TURBO DNase (Ambion) for 30 minutes at 37°C to eliminate the genomic DNA contamination. RNA (1 μg) was reverse transcribed with RevaTra Ace (TOYOBO) using oligo-dT primers in a 20 μL reaction volume. The cDNA was diluted with 80 μL distilled water, and 2 μL of the dilution was used for PCR assays.

Tissues and cells were homogenized in 1 mL of Sepazol and total RNA was extracted according to the manufacturer's instructions (Nacalai tesque). cDNA was synthesized from total RNA using RevaTraAce reverse transcriptase (Toyobo) and oligo dT primer. Real-time PCRs were performed to amplify fragments representing for the indicated mRNA expression using the Thermal Cycle DiceTM TP800 (Takara) and SYBR Premix Ex Taq (Takara). The primer sequences can be found in Supplementary Table S1.