Sybr premix dimereraser kit

Total RNA extraction and qRT-PCR analysis were performed as described by Guan et al. (55 (link)). Total RNA of the mycelia of the HN08 strain and mutants were extracted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China). cDNA synthesis was performed with TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). The expression levels of the target genes were quantified by qRT-PCR performed with an ABI7500 sequence detection system (Applied Biosystems, Waltham, MA, USA). Reactions were performed in a total volume of 10 µL using the SYBR Premix Dimer Eraser Kit (Takara, Beijing, China). All of the reactions were repeated in at least three independent pools in three sets of biological replicates. The primer sequences were listed in Table S1.

Use Reverse Transcription Kit (Takara, Japan) to convert RNA to cDNA according to the instructions. Using SYBR Premix Dimer Eraser Kit (Takara, Japan) to configure the experimental system, q-PCR amplification was carried out on the LightCycler@480II/96 (Roche) instrument, and the primer sequence was attached to Supplementary Table 1. The internal control used in the qPCR experiment was GAPDH. The operation procedure of the instrument is as follows: pre-denaturation, 1 cycle, 95°C, 30S, 20°C/S. PCR amplification, 40 cycles, 95°C, 5S, 20°C/S; 55°C, 30S, 20°C/S; 72°C, 30S, 20°C/S. Dissolution curve analysis, 95°C 0S, 20°C/S; 65°C, 15S, 20°C/S; 95°C, 0S, 0.1°C/S. After getting the Ct value at the end of the program, Graph Pad Prism6 is used to analyze the experimental results.

According to the manufacturer's instructions, Trizol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract the total RNA of cells and tissues, and the purity and integrity of the total RNA were tested. We reverse transcribed linc00887 into cDNA using the first strand synthesis system of superscript III (Thermo Fisher, Waltham, Ma, USA). Next, quantitative PCR was used to detect the specificity and amplification efficiency of primers. In the formal test, the total volume was 15 μL, including 6.5 μL sterile water, 7.5 μL SYBR Green PCR master mix, 0.5 μL upstream primer and 0.5 μL downstream primer. According to the manufacturer's instructions, U6 or GAPDH was taken as the control. Gene expression was identified by commercial kit SYBR ® premix dimer eraser Kit (Takara, Dalian, China). The PCR procedure was as follows: 95 °C 1 min, 1 cycle; 95 °C 20 s, 56 °C 1 s, 72 °C 15 s, 35 cycles. The relative quantification was determined by the 2^−ΔΔCt method. The primer sequences are as follows: linc00887 forward: 5′- ATC CAA GGA CTT GTG CTG GG-3′ and reverse: 5′-TGC TGA GCT GCT TCT TGG AA-3′; U6 forward: 5′-TGC GGG TGC TCG CTT CGG CAG C-3′ and reverse: 5′-CCA GTG CAG GGT CCG AGG T-3′; GAPDH 5′-TTG GTA TCG TGG AAG GAC TCA-3′ and reverse: 5′-TGT CAT CAT ATT GGC AGG TT-3′; FRMD6 forward: 5′-TGA CAC GCC ATA CAC AAG CT-3′ and reverse: 5′-CTT TGG CCT CAG ACT GAG CA-3′.

Total RNA was extracted from NSCLC cells or tissue samples by TRIzol (Invitrogen). PrimeScript RT reagent Kit With gDNA Eraser (TaKaRa) was used for reverse transcription, and the quantitative RT-PCR was performed by using SYBR Premix DimerEraser kit (TaKaRa) on the Roche LightCycler480 (Roche). Sequences of primers are shown in Table S2. The −2^ΔΔct method was used to analyze the data and the mRNA expression of β-actin was used as normalization control.

Total RNA was extracted using the RNAprep Pure Plant Kit (Tiangen, China) according to the kit instructions, and cDNA was synthesized using PrimeScript RT Master Mix (Takara, Japan) according to the kit instructions. PCR reactions were performed using the SYBR^® Premix DimerEraser™ kit (Takara, Japan) on the LightCycler 96 Real-Time PCR System (Roche, United States). Each PCR reaction was done three times. The relative expression levels of transcripts were measured and normalized using the comparative Ct (ΔΔCt) method compared to a reference gene NtEF1α gene (Kajikawa et al., 2017 (link)). qPCR primers were designed using Beacon Designer 8 software (Horejsh et al., 2005 (link)) and found in the additional file: Supplementary Table 7.