Evo m mlv rt premix for qpcr

RNA extraction and qRT-PCR analysis were carried out as described in our previous study (Zhang et al., 2021a (link)). Total RNA from cultured cells was extracted using SparkZol reagent (SparkJade, Shandong, China), and cDNA was synthesized with the Evo M-MLV RT Premix for qPCR (AG11706, Accurate Biotechnology, Hunan, China). The qRT-PCR analysis was carried out using the SYBR Green Premix Pro Taq HS qPCR Kit (AG11701, Accurate Biotechnology, Hunan, China) as directed by the manufacturer. Primers were synthesized by BioSune Co., Ltd. (Shanghai, China) and the sequences are shown in Table 1. The qRT-PCR was performed to evaluate the expression levels of the four lncRNAs (CTB-4E7.1, RP11-553A10.1, RP11-24N18.1, and PVRL3-AS1) using a LightCycler480 system (Roche Diagnostics, Switzerland). The lncRNAs expression was normalized to ACTB, and the relative expression level was determined by the 2^-∆∆CT method.
The study design and workflow of the present research is shown in Figure 1A. This study was approved by the Ethics Committee of Shandong Provincial Hospital (the ethics approval number is SZZJJ:NO.2021-419) and was carried out in accordance with relevant guidelines and regulations.

The total RNA from cells and skin tissue were extracted using AG RNAex Pro Reagent (Accurate Biology, Shanghai, China). cDNA was synthesized using a reverse transcription system kit according to the manufacturer’s instructions (Evo M-MLV RT Premix for qPCR, Accurate Biology, China). Quantitative real-time PCR was performed using the SYBR Green Premix Pro Taq HS qPCR kit (Accurate Biology, Shanghai, China) following the manufacturer’s protocol. PCR conditions were as follows: one step at 95 °C for 10 s, one step at 60 °C for 30 s, 72 °C for 5 s, and 2 s at 80 °C. All the above steps have 40 cycles. Gene expression levels were normalized to GAPDH and analyzed using the comparative cycle threshold (2^−ΔΔCt) method. Primer sequences for qPCR used in this study were listed in the Supplementary Table S1.

Total RNA was extracted using the SteadyPure Universal RNA Extraction Kit (Accurate Biotechnology Co., Ltd., Hunan, China; Cat# AG21017) in line with the instructions of the manufacturer. Later, the extracted total RNA was quantified and prepared into cDNA through reverse transcription using the Evo M-MLV RT Premix for qPCR (Accurate Biotechnology Co., Ltd., Hunan, China; Cat# AG11706). RT-qPCR was later performed using the QuantStudio 5 Real-Time System (Life Technologies) with SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology Co., Ltd., Hunan, China; Cat# AG11701). Each PCR procedure was run in duplicate. All gene expression data were calculated using the 2^–ΔΔCT method and normalized to β-ACTIN. The primer sequences for target genes are summarized in Supplementary Table 1.

Total RNA was collected from 66 pairs of breast cancer (BC) tissues and adjacent nontumor tissues frozen in liquid nitrogen using TRIzol reagent (Invitrogen, Waltham, MA, USA). Evo M-MLV RT Premix for qPCR (AG11706; ACCURATE BIOTECHNOLOGY, HUNAN, China) was carried out to reverse-transcribe RNA into complementary DNA (cDNA) according to the instruction. SYBR Green Premix Pro Taq HS qPCR Kit (ACCURATE BIOTECHNOLOGY (HUNAN) Co., Ltd., AG11701) was used to analyze RT-qPCR. The data were calculated using the 2−ΔΔCt method (Livak & Schmittgen, 2001 (link)). β-actin served as an internal standard control for mRNA expression. The primer sequences were as follows:
SKA3 Forward: 5′-TACACGAGCAAGAAGCCATTAAC-3′ and Reverse: 5′-GGATACGATGTACCGCTCAAGT-3′, β-actin, forward: 5′-TGGCACCCAGCACAATGAA-3′ and reverse: 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′.

The HiPure Total RNA Mini kit (Magen, China) was used to extract the total RNA of the PTR2 cells. The cDNA was reverse-transcribed from the RNA by Evo M-MLV RT Premix for qPCR (Accurate Biology, China). The Applied Biosystems StepOnePlusTM System was used to perform the quantitative real-time PCR (qPCR). The specific primers were designed to detect PRRSV and other genes (Table 2).

The RNA Prep Pure Plant Kit (Tiangen, Beijing, China) and Evo M-MLV RT Premix for qPCR (AG11706, Accurate Biotechnology, Hunan Co, Ltd.) were employed for the extraction of total RNA from L. chinense samples and subsequent cDNA synthesis, following the respective instructions. Total RNA from WT A. thaliana and transgenic plants was extracted using the KK Fast Plant Total RNA Kit (ZP405K, ZOMANBIO, Beijing). Subsequently, the Evo M-MLV RT Premix for RT-qPCR was utilized for cDNA synthesis.
We used RT-qPCR to detect the expression level of LcSPLs. A total of 10 μL reaction volume included 10 μM forward primer, 10 μM reverse primer, 0.2 μL 50× ROX, 5 μL 2× SYBR^® Green Pro Taq HS Premix (provided by SYBR^® Green Premix Pro Taq HS qPCR Kit (AG11701, ACCURATE BIOTECHNOLOGY)), 1 μL cDNA and up to 10 μL RNase-free water. For the amplification process and method of data analysis, refer to our previous study (Tu et al. 2022 (link)). LcACT97 was used as internal control gene for L. chinense, and AtACT2 was used as a reference gene for A. thaliana.

Total RNA was isolated from PBMCs using the RNAfast200 kit (Fastagen, China, #220011), according to the manufacturer’s instructions. cDNA was synthesized from total RNA using Evo M-MLV RT Premix for qPCR (ACCURATE BIOLOGY, China, #AG11706), according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was conducted using SYBR Green Premix Pro Taq HS qPCR Kits (ACCURATE BIOLOGY, China, #AG11701). To ensure the accuracy of qRT-PCR results, all RNA samples were extracted under consistent conditions, and equal amounts of RNA were used for reverse transcription to cDNA. Cycle threshold (CT) values of target genes were determined, and relative expression calculated using the 2^−ΔCT method. Data were normalized using the reference gene encoding β-actin, to correct for potential sample-to-sample variation. Primers used for qRT-PCR are listed in Table 3.Table 3

Primers used for qPCR in this study

Gene		Primer sequence (5ʹ → 3ʹ)
Human DRAM1	Forward	ATT GGT GGG ATG TTT CGG AAT GG
Human DRAM1	Reverse	TGA TGG ACT GTA GGA GCG TGT AC
Human LDHA	Forward	GTG TGC CTG TAT GGA GTG GA
Human LDHA	Reverse	GPC IAA CCA CCT GCT TGT GAA CCT
Human ACTB	Forward	CAT GTA CGT TGC TAT CCA GGC
Human ACTB	Forward	CTC CTT AAT GTC ACG CAC GAT