Sybr green kit

Total RNA was extracted from the indicated cells or mouse colon tissues with TRI reagent (Sigma) according to the manufacturer’s instructions. RNA samples were reverse-transcribed into cDNA with a GoScript^TM Reverse Transcription kit (Promega). The cDNA samples were amplified by real-time PCR with a SYBR Green kit (Toyobo) on an ABI 7900 HT Fast Real-Time cycler (Applied Biosystems). The expression of target genes was normalized to expression of housekeeping gene beta actin. In Figs. 2h and 6h, arbitrary units (AU) were introduced to normalize the difference between different batches of samples. Primers used were listed in Supplementary Table 3.

Total RNA of adult mouse SAN and atrium tissues was isolated using GenElute Single Cell RNA Purification Kit (Sigma, RNB300), total RNA of adult mouse ventricle, adult rabbit heart tissues, and neonatal rat cardiomyocytes were extracted using Trizol reagent (Thermo Fisher Scientific). Purified RNA was reverse-transcribed by the PrimeScript RT Reagent kit (Takara Bio, RR037A). Quantitative real-time PCR (qPCR) was performed using SYBR Green kit (TOYOBO CO, QPK-201) on the QuantStudio^TM 6 Flex system (Applied Biosystems; Thermo Fisher Scientific). GAPDH was used for endogenous control and the relative expression of mRNA level was quantified using 2^−△△CT method. The primers sequences are listed in Supplementary Data 7.

Mouse was transcardially perfused with normal saline to wash out blood. The brain was quickly removed from the skull and was chilled on ice for 10 min. Bilateral dissections of the BLA regions were performed according to a previous report (34 (link)). Coronal sections between Bregma −1 and Bregma −2.75 were isolated and verified under microscope. The sections were placed caudal side up. Cuts were then made in the lower-left and lower-right corners while avoiding any hippocampal tissue. Isolated BLA tissues were placed in the tube containing TRIzol reagent (Invitrogen, New York, NY, United States) and homogenized for 120 s. Total RNA was then extracted using chloroform and precipitated with isopropanol, followed by conversion to complementary DNA (cDNA) using SYBR Green kit (TOYOBO, Tokyo, Japan). The following primers were used: forward 5′-TGCCTCCTATTCTGTCTTATGC-3′ and reverse 5′-CTTTCAGGTCTTGACGCTCTAC-3′. PCR was performed using the ABI Q5 Real-Time PCR System (Applied Biosystems, Foster City, CA, United States). The relative IDO-1 gene expression was determined by normalization to the housekeeping gene (β-actin) and the IDO-1 gene in the control group using the 2−ΔΔ CT method.

Total cellular RNA was extracted by the RNeasy Mini Kit (Qiagen, Wuxi, China). A total of 600 ng RNA per treatment was reverse-transcribed by commercial SYBR green kit (TOYOBO, Japan). Quantitative real-time PCR (“qRT-PCR”) was performed through the ABI-7500 fast PCR system (Applied Biosystems, Shanghai, China) [67 (link), 68 (link)]. PP2A regulatory (B subunit) mRNA primers were reported early [45 (link), 69 (link)]. For miR analysis, miR was converted to cDNA from 600 ng of total RNA using the First-Strand Synthesis Kit (SABiosciences, Frederick, MD). miR-17-92 analysis was performed through qRT-PCR assay using the miR-17-92 primers (SABiosciences) [65 (link), 66 (link)]. miR-17-92 level was also normalized to GAPDH.

Total RNA of heart tissues and cell samples were extracted using a TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA synthesis was performed with a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan), and real-time PCR was performed using a SYBR Green Kit (Toyobo, Osaka, Japan) on the Roche LightCycler 480 System (Roche, Basel, Switzerland). The primer sequences are shown as in Table 1.