Stepone software program

Cyanobacterial cultures in liquid medium were incubated for 72 h, followed by centrifugation, immediate extraction of RNA, and freezing with liquid nitrogen. The RNA from each sample was quantified and used to synthesize cDNA using the Hiscript II Q RT Supermix for qPCR (+gDNA wiper). The obtained cDNA was diluted 100-fold and then used as the template for qRT-PCR. The qRT-PCR mixtures were prepared with the ChamQ Universal SYBR qPCR master mix (Vazyme, Nanjing, China) according to the manufacturer’s instructions. The qRT-PCRs were then performed using the StepOne real-time PCR system (Applied Biosystems, CA, USA). The rnpB gene encoding RNase P subunit B was used as a housekeeping gene. Three technical replicates were used for each sample. The qRT-PCR data were analyzed using the StepOne software program (Applied Biosystems) and the threshold cycle (2^−ΔΔCT) method (26 (link)).
Sixteen genes were selected to assess the reliability of RNA-seq expression data with qRT-PCR, using the primers shown in Table S6. Three technical replicates were used for each sample. Correlations between RNA-seq and qRT-PCR were assessed via Pearson correlation coefficients using the Excel software program. Correlation coefficients of >0.8 were used to identify reliable RNA-seq expression data.

After 6 h of stimulation by the indicated hormone (10 nM, 500 nM or 50 μM of T1AM, 100 nM of ET-1, and 20 μM of PE), each dish was snap frozen. Where indicated, NRCM were first pretreated with 500 nM of AZD6244 (Cayman Chemical) or 10 μM of H-89 30 min prior to the indicated hormone stimulation. Total RNA was extracted from the frozen cells using TRIzol reagent (Invitrogen) and a quantitative real-time PCR was performed using a StepOnePlus Real-time PCR System and the StepOne Software program (Applied Biosystems), as described previously^{15 (link),37 (link)}. The real-time PCR protocol consisted of one cycle at 95 °C for 20 s followed by 40 cycles at 95 °C for 1 s and 60 °C for 20 s using the primers for NPPB (Applied Biosystems, Rn00580641_m1) and GAPDH (Applied Biosystems, Rn01775763_g1). The transcriptional levels were determined using the ΔΔCt method with normalization to GAPDH^{15 (link)}.

Total RNA was extracted from frozen NRCMs, cardiac fibroblasts and frozen heart tissue using TRIzol reagent (Invitrogen), and quantitative real-time PCR was performed using a StepOnePlus Real-time PCR System and the StepOne Software program (Applied Biosystems), as previously described.⁷ (link) The RT‒PCR protocol consisted of one cycle at 95°C for 20 s followed by 40 cycles at 95°C for 1 s and 60°C for 20 s using the primers for URAT1 (Applied Biosystems, Mm01244861_m1 and Rn01479630_g1), TNFα (Applied Biosystems, Mm00443258_m1 and Rn99999017_m1), MCP1 (Applied Biosystems, Mm00441242_m1 and Rn00580555_m1), IL-1β (Applied Biosystems, Mm00434228_m1 and Rn00580432_m1), CD68 (Applied Biosystems, Mm00432403_m1), NLRP3 (Applied Biosystems, Mm00840904_m1) and GAPDH (Applied Biosystems, Mm03302249_g1 and Rn01775763_g1). The transcriptional levels were determined using the ΔΔCt method with normalization to GAPDH.

Total RNA was extracted from the frozen tissues using TRIzol reagent (Invitrogen) and a quantitative real-time PCR was performed using a StepOnePlus Real-time PCR System and the StepOne Software program (Applied Biosystems), as described previously^{9 (link),55 (link)}. The real-time PCR protocol consisted of one cycle at 95 °C for 20 s followed by 40 cycles at 95 °C for 1 s and 60 °C for 20 s using the primers for UCP1 (Mm01244861_m1; Applied Biosystems). The transcriptional levels were determined using the ΔΔCt method with normalization to GAPDH (Mm99999915_g1; Applied Biosystems).

Total RNA was isolated from the ipsilateral hemisphere after HI using the RNeasy Kit (QIAGEN) and measured using a NanoDrop (Thermo Scientific). All the samples had a nucleotide ratio (A260: A280) within the range of 1.9–2.1. A total of 1 μg of extracted RNA underwent reverse transcription into cDNA using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol. Quantitative PCR (qPCR) was performed using the Step-One-Plus Real-Time PCR machine (Applied Biosystems) with Power SYBR Green PCR Master Mix (Applied Biosystems) and the primers presented in Table S2. The set-up condition was initial denaturation at 95 °C for 5 min, subsequent denaturation at 95 °C for 5 s, annealing at 60 °C for 10 s, and elongation for 30 s for a total of 40 cycles. The cycle time values were normalized to the β-actin of the same sample. The mRNA expression levels were calculated using the delta-delta CT method as fold changes compared to the DTA⁻ sham samples at P13. For pre-HI insult assessments, DTA⁺ samples were compared with DTA⁻ samples at P10 after tamoxifen administration at P8 and P9. All samples were examined in triplicate. The aforementioned analyses were performed using the StepOne software program (version 2.3, Applied Biosystems).