Evo m mlv rt mix kit with gdna clean

Collect the blood samples collected by the patients within 24 h after admission from the laboratory department. PBMCs were isolated from the blood samples using density gradient centrifugation with Ficoll-Hypaque which purchased from 4 A Biotech Co., Ltd according to the manufacturer’s instruction. Total RNA was extracted from PBMCs, and genomic DNA (gDNA) was removed for reverse transcription. Trizol reagent and Evo M-MLV RT Mix Kit with gDNA clean were purchased from Accurate-Biology, Hunan, China.

For qRT-PCR, RNA was extracted using a Trizol Total RNA Extraction Kit (Sangon Biotech, Shanghai, China) and reverse transcribed using an Evo M-MLV RT Mix Kit with gDNA Clean (Accurate Biotechnology, Hunan, China). The primers are listed in Supplementary Table 1. qRT-PCR was conducted using a BioEasy Master Mix (SYBR Green) Kit (Bioer, Hangzhou, China) and a C1000 TouchChihermal Cycler system (Bio-Rad). All tested transcripts were normalized to the reference gene TLF, and relative transcript levels were calculated according to the 2^-ΔΔCt method (Tian et al., 2016 (link)). Three biological and technical replications were performed.

Nine DEGs were randomly selected for qRT-PCR analysis to verify the accuracy of the sequencing results. TRIzol Total RNA Extraction Kit (Sangon Biotech, Shanghai, China) was used to extract RNA. RNA was reverse-transcribed using an Evo M-MLV RT Mix Kit with gDNA Clean (Accurate Biotechnology, Hunan, China). The primers are shown in Supplementary Table 2. qRT-PCR was performed using a BioEasy Master Mix (SYBR Green) Kit (Bioer, Hangzhou, China) and a C1000 TouchChihermal Cycler system (Bio-Rad, Hercules, CA, USA). The reference gene Actin was used to normalize all transcripts tested. Relative transcript levels were calculated using the 2^−ΔΔCt method with three biological replications (Livak and Schmittgen, 2001 (link)).

Total RNA of tissues or cells was extracted using the TRIzol reagent (Life Technologies, Carlsbad, CA, USA) based on the manufacturer’s instructions. Agarose gel electrophoresis was employed to assess the integrity of the RNA. A 28S band with an intensity approximately twice that of the 18S band was used for a preliminary assessment of relatively intact total RNA. The quality and concentration of the RNA were detected using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and 1 μg of RNA was reverse transcribed into cDNA using an Evo M-MLV RT Mix Kit with gDNA Clean (Accurate Biology, Hunan, China). A SYBR Green Kit (Accurate Biology) was used for the qPCR step of the qRT-PCR protocol. Relative mRNA expression levels were analyzed using the 2-ΔCT method [26 (link)] and standardized against the expression of ACTB (encoding beta actin). All primer sequences used in this study are listed in Additional file 2: Table S2 and S3.

Total RNA was extracted from leaves of U. rhynchophylla using the SteadyPure Plant RNA Extraction Kit (Accurate Biology, Hunan, China). The purity and concentration of RNA were measured by Micro Drop (BIO-DL, Shanghai, China), and the RNA integrity was checked on 1% agarose gels. Total RNA (500 ng) was used for reverse transcription with Evo M-MLV RT Mix Kit with gDNA Clean (Accurate Biology, Hunan, China) to remove genomic DNA contamination for qPCR in a 20 μL volume according to the manufacturer’s instructions.
The pBI121-GFP vector, carrying a CaMV 35S promoter, was selected for characterizing the transformation efficiency of U. rhynchophylla protoplasts. To verify the feasibility of detecting the subcellular location of a U. rhynchophylla protein, the complete coding sequences of UrWRKY37 with no stop codons including the restriction enzyme sites Ndel and SmaI were amplified using the primers 5′-GACAGTGACATATCCAAGAGACAGA-3′ (forward primer) and 5′-GCCAGCGATAGCCATCATTTAC-3′ (reverse primer) and cloned into the pBI121-GFP vector, establishing the expression plasmid pBI121-UrWRKY37-GFP. Plasmid DNA was extracted using the Plasmid MaxiPrep Kit (endotoxin-free) (Coolaber, Beijing, China). The DNA concentration was at least 500 ng/μL. All plasmid DNA were stored at −20 °C after confirmation.