Evo m mlv rt kit

The total RNA from 60 PDAC samples (Ruijin cohort) was extracted using TRIzol reagent (Invitrogen, United States) and reverse-transcribed using an Evo M-MLV RT Kit (Accurate Biology, China). Real-time PCR was performed with an ABI 7900 instrument using ChamQ SYBR qPCR Master Mix (Vazyme, China). Quantitation was performed in triplicate. mRNA expression was calculated using the 2^–ΔΔCT method and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers for the amplified mRNAs are summarized in Supplementary Table S1.

Total RNA was isolated from liver tissues using TRIzol reagent (Accurate Biology, Hunan, China). Subsequently, RNA samples were reverse transcribed into cDNA using the Evo M-MLV RT Kit (Accurate Biology, Hunan, China). The cDNA was used as the template for the real-time quantitative PCR (RT-qPCR) reaction. The primers were synthesized by Sangon (Shanghai, China). RT-qPCR was performed using SYBR^® Green Premix Pro Taq HS qPCR Kit (Accurate Biology, Hunan, China) according to the manufacturer’s instructions. RT-qPCR is carried out in the C1000Touch^™ Thermal cycle System (Bio-Rad, CA, United States). The relative expression for a particular gene was calculated using the method of 2^–ΔΔC. The primer sequences were listed in Table 1.

Total RNA samples from cultured GCTB cells or mouse tumors were prepared using the total RNA extraction kit (#15596018; Invitrogen, Guangzhou, China), as per manufacturer’s instructions. RNA concentrations were measured using NanoDrop 2000 instrument (Thermo Fisher Scientific, United States). The cDNA for quantitative reverse transcription polymerase chain reaction (qRT-PCR) was subsequently synthesized from 1 μg total RNA using Evo M-MLVRT kit (#AG11706; Accurate Biotechnology, Hunan, China), according to the manufacturer’s instructions. Then, the qRT-PCR assay was performed using the LightCycler 480 SYBR Green I Master Kit (#4887352001-1; Roche, Guangzhou, China), as per the manufacturer’s instructions. The final expression levels of mRNAs were calculated by the standard 2^–ΔΔCt method based on at least three biological replicates. The primer sequences used for quantitation are listed in Table 1.

RNA was extracted from the whole right hippocampus tissue using the TRIzol Reagent as described previously. Reverse transcription was performed using Evo M-MLV RT Kit (AG11711, Accurate Biology), and SYBR® Green Premix qPCR Kit (Thermo Fisher, Applied Biosystems QuantStudio 5) was used to perform qRT-PCR (AG11718, Accurate Biology). The primer sequences are listed in Table S2. All relative gene expression analyses were performed using the 2^−ΔΔCt and were normalized to GAPDH as the reference gene.

The AG RNAex Pro Reagent (AG21102, Accurate Biology, Changsha, China) was used to extract total RNA from 55 fresh hepatocellular carcinoma tissues and part of paired normal liver tissues according to the manufacturer’s instructions. Before reverse transcription to cDNA, genomic DNA is eliminated by treatment for 2 min at 42°C with gDNA Clean Reagent. Evo M-MLV RT Kit (AG11705, Accurate Biology, Changsha, China) was used to synthesize the complementary RNA. The SYBRR Premix Pro Taq HS qPCR Kit (AG11708, Accurate Biology, Changsha, China) was utilized to achieve real-time quantification. The 2^-△△CT approach was applied to assess the relative expression levels of target genes, which were normalized by GAPDH. The PCR primers are listed in Supplementary Table S6.

The primary hepatocytes were lysed using the AG RNAex Pro regent (Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China) for 5 min at room temperature. Then, total RNA was extracted from the lysis using a SteadyPure RNA Extraction Kit with reference to the protocols provided by Accurate Biotechnology. Agarose gel electrophoresis was performed to confirm the quality of RNA samples with the purity determined by a Nanodrop UV spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Then, the first-strand complementary DNA (cDNA) was obtained by reverse transcription from 1 μg of total RNA using an Evo M-MLV RT Kit (Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China). To quantify gene expression, the real-time PCR (RT-PCR) was conducted under the Bio-Rad CFX96 platform (Bio-Rad, Berkeley, CA, USA) using the SYBR Green Pro Taq HS premix acquired from Accurate Biotechnology. The 2^−∆∆Ct method was adopted to calculate the relative transcription with elongation factor 1α (ef1a) used as the housekeeping gene. The high expression stability of ef1a in M. amblycephala was demonstrated in a recent study [32 (link)]. Moreover, the sequences of primers used in the current study are listed in Table 1.