Evo m mlv rt master mix

Total RNA was extracted from the cells using SteadyPure RNA Extraction Kit (Accurate Biotechnology Co., Ltd.) according to the manufacturer’s instructions. The cDNA synthesis was performed using 5X Evo M-MLV RT Master Mix (Accurate Biology, AG11706). RT-PCR experiments were conducted using 2X SYBR Green Pro Taq HS Premix (Accurate Biology, AG11701). Using Beta-actin as a control. The relative expression of the target genes relative to the control was calculated according to the 2−ΔΔCT formula. Each experiment was performed in triplicate.

Total RNA from tumor tissues of 79 PDAC patients who had been treated in our cancer center was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA from cells was extracted using a SteadyPure Universal RNA Extraction Kit (Accurate Biotechnology, China). cDNA was obtained by reverse transcription polymerase chain reaction (RT-PCR) using 5x Evo M-MLV Rt Master Mix (Accurate Biotechnology, China). Quantitative real-time polymerase chain reaction (qPCR) was performed using a QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific, USA). The sequences of the PDGFRA primers used were as follows: human PDGFRA, 5′-TGGCAGTACCCCATGTCTGAA-3′ (forward primer) and 5′-CCAAGACCGTCACAAAAAGGC-3′ (reverse primer) and human GAPDH, 5′-GGAGCGAGATCCCTCCAAAAT-3′ (forward primer) and 5′-GGCTGTTGTCATACTTCTCATGG-3′ (reverse primer).

Total RNA was isolated from mouse liver tissues with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and was converted to cDNA with the Evo M-MLV RT Master Mix (Accurate Biotechnology, Changsha, China). A quantitative real-time PCR (qRT-PCR) was performed on Bio-Rad CFX 96 (Bio-Rad Laboratories, Hercules, CA, USA) with SYBR^® Green Pro Taq HS Premix II (Accurate Biotechnology). The relative mRNA expression levels were assessed using the 2^−ΔΔCq method and normalized by the Actb (used as a control gene). The primers were designed from IDT DNA Technology (Coralville, IA, USA) and synthesized by Shenggong Bioengineering Co., Ltd. (Shanghai, China). The primer sequences used for the PCR are shown in Table S1.

The extracted total RNA from mouse aortas and livers (n = 10 per group) in bulk RNA‐seq was also used for qPCR. mRNA was reversely transcribed to cDNA using an Evo M‐MLV RT Master Mix (Accurate Biotechnology, Hunan, China). qPCR was performed using a 5× Priescript RT Master Mix and an SYBR Green Realtime PCR Master Mix (Takara, Shiga, Japan) with an Applied Biosystems ViiA 7 Real‐Time PCR (Thermo Fisher Scientific). The 2^−ΔΔCT method^[104] was adopted to calculate the tested genes which were normalized by β‐actin. The sequence of mRNA primers (synthesized by Tsingke) used in this study was presented in Table S7 of the Supporting Information.

We extracted total RNA from collected KIRC tumor tissues and paracancerous normal tissues using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). Next, we reverse-transcribed the pre-extracted RNA into cDNA using EvoM-MLVRT master mix (Accurate Biotechnology). We then mixed the reagents for qRT-PCR detection according to the manufacturer’s instructions of the SYBR^® Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology). The above process was carried out in strict accordance with the manufacturer’s instructions.

To extract total RNA, the rat renal tissues (50 mg) or cultured HK-2 cells were homogenized in 500 μL TRIzol^® reagent (Invitrogen, Waltham, MA, USA). The total RNA pellet was dissolved in diethylpyrocarbonate (DEPC) water. The quality and quantity of total RNA were detected using ultraviolet absorption (optical density, 260 nm/280 nm) with a NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). An equal amount of total RNA (500 ng) was reverse transcribed to cDNA using Evo M-MLV RT Master Mix (Accurate Biotechnology, Hunan, China). Quantitative real-time PCR (qRT-PCR) was performed on Bio-Rad CFX 96 (Bio-Rad, Hercules, CA, USA) with SYBR^® Green Pro Taq HS Premix II (Accurate Biotechnology). The relative mRNA expression levels were assessed using the 2^−ΔΔCq method and normalized using gene GAPDH (used as an internal control gene). The primers were designed from IDT DNA Technology (Coralville, IA, USA) and synthesized by Sangon Biotech, Co., Ltd. (Shanghai, China). The primer sequences used for PCR are shown in Table S1.

The total RNA from the culture was isolated with the SteadyPure Universal RNA Extraction Kit (AG21017, Accurate Biology, China). Evo M-MLV RT Master Mix (AG11706, Accurate Biology, China) was used for cDNA synthesis through reverse transcription. SYBR Green Pro Taq HS Premix (04707494001, Roche, Switzerland) was used for real-time quantitative PCR (RT-qPCR) according to the manufacturer’s instruction on CFX96 Touch (Bio-rad) (n = 3). The primer sequences are listed in Supplementary Table 1.