Evo m mlv reverse transcription kit

Total RNA was extracted using Trizol reagent (accurate biotechnology CO., LTD, China) according to the manufacturer's instructions and total RNA concentration measured. The E-vo M-ML V Reverse Transcription Kit (Accurate Biotechnology CO., LTD, China) was used to reverse transcribe RNA into cDNA. The real-time PCR kit was ordered from Takara, and the real-time PCR instrument (lightCycler 2.0) was purchased from Roche. A standard curve was drawn based on the Cp value obtained from the reaction and the amplification efficiency of the gene calculated. To analyze the gene expression, real-time quantitative PCR was performed using an ABI Prism 7300 instrument (Applied Biosystems, Foster City, CA). The total reaction volume was 10 μL, which contained 1 μL cDNA. StepOnePlus instrument (Applied Biosystems) was used for PCR amplification. The primer sequences used for amplification of genes are listed in Table 1.

All clinical samples were resuspended in 0.9% sodium chloride solution (Shanghai Xin Yu Biotech Co., Ltd). RNA was extracted from 200 μL homogenized sample using Magnetic bead method nucleic acid extraction kit (Hangzhou Bioer Technology Co., Ltd) following the manufacturer's instructions. An Evo M-MLV Reverse Transcription Kit (Accurate Biotechnology Co., Ltd.) was used to reverse transcribe the extracted RNA into cDNA according to the manufacturer's instructions. cDNA products were stored at −20°C until further study.

Total RNA was extracted from plant samples using a SteadPure Plant RNA Extraction Kit (Accurate Biotechnology, Hunan), according to the manufacturer’s instructions. cDNA was synthesized in 500 ng total RNA using an Evo M-MLV reverse transcription kit (Accurate Biotechnology, Hunan). Primer design of genes was carried out using the online website https://www.genscript.com/tools/real-time-pcr-taqman-primer-design-tool (accessed on 9 May 2023) (see Table S6). The system and procedure of RT-PCR referred to Zhang et al. [75 (link)]. In brief, the total RT-PCR system amounted to 10 µL, including 5 µL of 2X SYBR Green Pro Taq HS Premix*, 1 µL of cDNA, 0.8 µL each of upstream and downstream primers (10 µmol/L), and ddH₂O. The RT-PCR reaction procedure was as follows: step 1, 95 °C for 30 s; step 2, 95 °C for 5 s, 60 °C for 30 s, 72 °C for 10 min for 40 cycles, 65 °C for 5 s, 95 °C for 5 s; 3 repetitions. The relative expression of genes was calculated by the 2^−ΔΔCt method.

Total RNA was isolated using the RNAiso reagent (TAKARA). An equal concentration of obtained RNAs (500 ng) was reverse transcribed to cDNA through the Evo M-MLV Reverse Transcription kit (Accurate Biology) according to the manufacturer’s instructions. The synthesized cDNAs were subjected to real-time quantitative PCR using the SYBR Green Master kit (Yeasen). Specific primers (Table 1) for CHOP, GRP78, ATF4, XBP1s, XBP1u, CDV-N, and GAPDH were utilized for amplification. Quantitative PCR (qPCR) was conducted on StepOnePlus Real-Time PCR System and the relative mRNA levels were calculated by the 2^−ΔΔCT method. To detect XBP1 mRNA splicing, the primers XBP1u-F2 and XBP1u-R in Table 1 were used to amplify XBP1 mRNAs from synthetized cDNAs by PCR using ApexHF HS DNA Polymerase FS Master Mix (Accurate Biology) according to the manufacturer’s instruction. The examination of XBP1 mRNA splicing was performed by the restriction endonuclease Pst I (TAKARA) only digesting the XBP1u isoform as described in previous literature (16 (link)).

Cellular or tissue RNAs were extracted using TRI reagent (T9424; Merck, Darmstadt, Hesse, Germany). Reverse transcription was performed using 500 ng of total RNA with the Evo M-MLV Reverse Transcription Kit (AG11706; Accurate Biology, Hunan, China). Reactions were performed in technical and biological triplicate using a 96-well format on an ABI Step One Plus system, using Hieff qPCR SYBR Green Kit (11201; YESEN Biology, Shanghai, China). The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) conditions were 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Gene expression was normalized to β-actin or 36B4, and relative expression was calculated using the 2–(ΔΔCt) method. Primers were used at 0.5 μM, and their sequences are listed in Supplementary Table 6. PCR efficiency was optimized and melting curve analyses of products were performed to ensure reaction specificity.

Fin DNA was collected with MagPure Tissue DNA DA Kit and the corresponding DNA extractor (Magen, Guangzhou, China). RNA was collected with HiPure Universal RNA Mini Kit (Magen, Guangzhou, China), and DNA synthesis with Evo M-MLV reverse transcription kit (Accurate Biology, Guangzhou, China). In addition, the concentration and mass of DNA and RNA were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and a 1% agarose gel, separately.

Total RNA was isolated from lung tissue samples using TRIzol reagent (Solarbio, Beijing, China). The concentration and purity of extracted RNA were detected by spectrophotometer. The extracted RNA was reverse transcribed into cDNA using an Evo M-MLV Reverse Transcription Kit (Accurate Biology, Changsha, China). SYBRR Premix Ex TaqTM II (TaKaRa, Beijing, China) was used for quantitative real-time fluorescence analysis. The primers were synthesized by Shanghai Sangong Biological Company (Table 1). mRNA expression was calculated by the 2^−ΔΔCt method, and β-actin was the internal reference gene.