Evo m mlv rt kit with gdna clean for qpcr 2

The total RNA from potato was extracted using the TRIGene Total RNA Extraction Reagent (GenStar, Shenzhen, China, P118-01), and then Evo M-MLV RT Kit with gDNA Clean for qPCR II (Accurate Biology, Changsha, China, AG11711) was used to perform reverse transcription, according to the manufacturer’s instructions. The design of StPAL gene-specific primers for quantitative real-time PCR (qRT-PCR) analysis was investigated using the Primer Premier 6 software and NCBI. The ef1α gene was used to normalize the results (Table A4). The qRT-PCR process was performed on the Q7 Real-Time PCR System. In qRT-PCR experiments, the following thermal cycling conditions were applied: initial activation of 94 °C for 2 min, then 40 cycles of 94 °C for 15 s, 60 °C for 15 s, and 72 °C for 30 s. The relative expression levels were calculated using the comparative 2^−∆∆CT method [65 (link)].

The LABGENE^TM Plant RNA Isolation Kit (Cat. No. LB1111, LABGENE Biotechnology Co., Ltd., Chengdu, China) was applied to isolate the total RNA of all the samples. RNA concentration and quality were measured by using NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, United States) and gel electrophoresis respectively. An amount of 1.0 μg good-quality RNA was used for cDNA synthesis by the Evo M-MLV RT Kit with gDNA Clean for qPCR II (Code No. AG11711, Accurate Biotechnology Co., Ltd., Changsha, China). Then, the cDNA was stored at −20 °C for further use. In this study, we used the premier 5.0 software to design premiers for real-time quantitative PCR (qRT-PCR), showing the sequences in detail in Table S4. qRT-PCR was performed on a CFX96 Real-time PCR Detection System with the SYBR^® Green Premix Pro Taq HS qPCR Kit (Cat No. AG11701, Accurate Biotechnology Co., Ltd., Changsha, China). The qRT-PCR program was set as follows: 95 °C for 30 s, and 40 cycles of 95 °C for 5 s, 58 °C for 20 s, and 72 °C for 30 s. Three biological replicates and technical replicates were performed in all experiments. The 2^−ΔΔCT method was used to analyze the relative expression levels [68 (link)]. Unigene.16454 and Unigene.16459 were used as the internal reference genes for different tissues, and IDH and SDP were used as reference genes for different hormone treatments [69 (link)].

RNA Express Total RNA Kit M050 (New Cell & Molecular Biotech) were used to extract RNA from TAMs, M1-NTRMs and THP-1 cells. PARIS Kits (Invitrogen) were applied to separate RNA fractions of THP-1 cells from nucleus and cytoplasm. cDNA was synthesized by using Evo M-MLV RT Kit with gDNA Clean for qPCR II AG11711 (Accurate Biotechnology(Human)Co.,Ltd.). SYBR Green Premix Pro Taq HS qPCR Kits AG11721 (Accurate Biotechnology(Human)Co.,Ltd.) was used for quantitative real-time PCR assays. GAPDH Total RNA from TAMs, monocytes from patients’ peripheral blood and cell lines used in this study was extracted in TRIzol (Invitrogen, USA). RNA in nucleus or cytoplasm was extracted with a PARIS Kit (Invitrogen, USA). We reverse transcribed synthesized cDNA by using HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme). GAPDH was designated as a positive control of internal mRNA and PACERR. U6 was used as positive control of internal miRNA and PACERR which was in nucleus. The related primers are given in Additional file 4: Table S4.

Total RNA was extracted by Trizol (Invitrogen, United States). According to the manufacturer’s protocol affiliated with Evo M-MLV RT Kit with gDNA Clean for qPCR II (Accurate Biology, China), circRNA was reverse transcribed to complementary cDNA and then quantified by SYBR Green Real-time PCR Master Mix (Accurate Biology, China). β-actin was used as the endogenous control, detailed information about these primers was provided in Supplementary Table 1. The expression levels of circRNAs were normalized to β-actin, and the relative expression levels of circRNAs were shown by the value of the 2^–ΔΔCt.

Total RNA was isolated from bacterial culture using EASYSpin Plus Bacterial RNA kit (Aidlab Biotechnologies, China) according to the manufacturer’s guide. Genomic DNA was removed and cDNA was synthesized using Evo M-MLV RT Kit with gDNA Clean for qPCR II (Accurate Biology, China). qRT-PCR was carried out using SYBR Green Pro Taq HS qPRC Kit (Accurate Biology, China) with the QuantStudio 1 system (Applied Biosystems). To normalize total RNA quantity of different samples, constitutively transcribed gene rpoD was used as a reference control. The relative amount of mRNA level was calculated using the ΔΔCt method. Three independent biological samples with two technical repeats for each sample were performed. The RACE experiment was carried out using SMARTer RACE 5′/3′ Kit (Clontech) according to the manufacturer’s instructions.

Total RNA was extracted from human liver tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The concentration and purity of RNA were measured using a NanoDrop 2000 apparatus (Thermo Fisher Scientific, Waltham, MA, USA). The RNA integrity was evaluated by the ratio of 28S : 18S after denaturing formaldehyde agarose gel electrophoresis. cDNA was synthesized with Evo M-MLV RT Kit with gDNA Clean for qPCR II (Accurate Biology, Hunan, China) according to the manufacturer's instructions. qRT-PCR was performed using a LC480 real-time PCR detection system (Roche, Basel, Switzerland) with GoTaq qPCR Master Mix (Promega, Madison, Wisconsin, USA). All primers used for qRT-PCR were designed using Beacon Designer 7.0 and synthesized by BGI Tech (Shenzhen, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as internal control. The sequences of all primers used are listed in Supplementary table 3.