Reverse transcription

VEGF and bFGF expression levels in the liver tissue were detected using a TRIzol kit (Shanghai Biological Engineering Technology Service Co., Ltd., Shanghai, China); reverse transcription (RT)-PCR (Takara, Shiga, Japan); two-step RT-PCR kit; and DL 1,000 DNA marker (Takara Biotechnology Co., Ltd., Dalian, China). The primers used for qPCR were as follows: VEGF forward, 5′-ACCTCACCAAAGCCAGCACA-3′ and reverse, 5′-GGC ATGGTGGTGACATGGTT-3′ (amplification product, 536 bp); bFGF forward, 5′-ACACGTCAAACTACAACT CCA-3′ and reverse, 5′-TCAGCTCTTAGCAGACATTGG-3′ (amplification product, 243 bp). qPCR was performed using the Mx3000P qPCR System (Stratagene, La Jolla, CA, USA). The cDNA was then used for qPCR in 20 μl SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd). qPCR was performed under the following conditions: 5 min at 95°C, 40 cycles of 30 sec at 95°C, 30 sec at 60°C, and 1 min at 72°C. All results were normalized against β-actin amplification. CT values for triplicate reactions were averaged and relative expression was determined using the comparative CT method.

RNA was extracted from colon tissue and Caco-2 cells by TRIzol, and reverse transcription (Takara, Japan) and real-time PCR (Vayme, China) were performed according to the instructions of the kit. All primer sequences were synthesized by Sangon Biothen Co., Ltd. (Shanghai, China), and the primer sequences are shown in Table S1. All qRT-PCR analyses were carried out with a 7500 real-time PCR system (Applied Biosystems). The amplification protocol was as follows: 95 °C for 30 s, 40 cycles 95 °C for 10 s, 60 °C for 35 s, 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s. The relative fold change in expression was finally calculated using the 2^−ΔΔCT method. All samples were assayed in triplicate and expression levels of mRNA were normalized using the endogenous control β-actin [35 (link)].

Zebrafish were treated with CsA for 72 h, 40 juveniles were taken from each group to extract total RNA, and 1 μg total RNA was used for reverse transcription (Takara). The cDNA obtained was used for qPCR experiments on the ABI Step One Plus RT-PCR system (Applied Biosystem, CA, United States), and the experiments were repeated for 3 times. The expression of cardiac related developmental genes (GATA4, Nkx2.5, vmhc, kl2a), apoptotic related genes (p53, mdm2, bax) and Wnt signaling pathway related genes (β-catenin, lef1, axin2) were analyzed, and 2^-△△Ct formula was used to calculate the results. Primers were obtained from Thermo Fisher.

The total RNA samples from rat livers were extracted according to the manufacturer’s protocol (TaKaRa Bio, Otsu, Japan). The mRNA levels of sterol regulatory element-binding protein 2 (SREBP2), cholesterol 7α-hydroxylase (CYP7A1), liver X receptor (LXR), farnesoid X receptor (FXR) and low-density lipoprotein receptor (LDLR) were measured as described previously [50 (link)]. Total RNAs were extracted with TRIzol (Thermo Scientific, Wilmington, DE, USA), and cDNAs were synthesized by reverse transcription (TaKaRa Bio, Otsu, Japan). The quantitative real-time polymerase chain reaction (RT-qPCR) used the SYBR Premix Ex TaqII (TaKaRa Bio, Otsu, Japan) and the appropriate primers. The primer sequences which were obtained from Sangon Biological Engineering (Shanghai, China) are shown in Table 4. The thermocycler conditions used were 95 °C for 30 s, followed by 45 cycles of 95 °C for 5 s and 60 °C for 30 s.

Total RNA was prepared with the TRIzol reagent (Invitrogen) and reverse transcription (Takara) was performed following the manufacturer's protocol (Takara). Using forward and reverse primers, RT-QPCR assay was performed in the presence of GoTaq^® qPCR Master Mix (Promega) in accordance with the manufacturer's instructions. The relative quantitation of gene mRNA expression was normalized to β-actin in the same sample. The sequences of the primers were as follows:
Human β-actin: forward 5′-CCAACCGCGAGAAG ATGA-3′, reverse 5′- CCAGAGGCGTACAGGGATAG-3′; Human AEG-1: forward 5′-AACAGCAAAGCAGCCAC CAGAG-3′, reverse 5′-CAGGAAGGAAGGCTGGAA GAGTG-3′; Human MMP-9: forward 5′- TTGACAGCG ACAAGAAGTGG-3′, reverse 5′-GCCATTCACGTCGT CCTTAT-3′; Human IL-6: forward 5′-AAGCCAGAGCTG TGCAGATGAGTA-3′, reverse 5′-TGTCCTGCAGCCAC TGGTTC-3′; Human IL-12: forward 5′-ACCCTGACCA TCCAAGTCAAA-3′, reverse 5′-TTGGCCTCGCATC TTAGAAAG-3′; Human TNF-α: forward 5′-CCCAGGG ACCTCTCTCTAATC-3′, reverse 5′-GCTGGTTATCTCT CAGCTCCA-3′; Human TGF-β: forward 5′-CAACA ATTCCTGGCGATACC-3′, reverse 5′-GAACCCGTTGA TGTCCACTT-3′; Human IL-10: forward 5′-GACTTTA AGGGTTACCTGGGTTG-3′, reverse 5′-TCACATG CGCCTTGATGTCTG-3′.

Total RNA was extracted from tissue using Trizol reagent (Invitrogen). RNA was precipitated with isopropanol and dissolved in diethyl pyrocarbonate-treated distilled water. First-strand cDNA was generated with oligo dT-adaptor primers by reverse transcription (TaKaRa). Specific primers were designed using qPrimerDepot (http://mouseprimerdepot.nci.nih.gov, Table S1). The real-time reverse transcription polymerase chain reaction (RT-PCR) reaction systems had a final volume of 10 μl and contained 10 ng of reverse-transcribed total RNA, 200 nM forward and reverse primers, and a PCR master mix. RT-PCR was performed in 384-well plates using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). reverse transcription and PCR were performed using a One-Step RT-PCR kit (Invitrogen). The PCR products were separated by electrophoresis on 2% agarose gels, followed by staining with ethidium bromide.