Transstart top green qpcr supermix kit

Total RNA was extracted from ~60 flies aged 5–8 days using TRIzol reagent (Invitrogen) and then reverse transcripted by the PrimeScript^TM RT Master Mix kit (Takara, RR036A). Quantitative PCR analysis was then performed using TransStart Top Green qPCR SuperMix kit (TransGen, AQ131-03) in the Applied Biosystems 7900HT Fast-Time PCR system. The sequences of primers used to detect shmt and actin42a (endogenous control) RNA are as follows:
shmt-F: 5′-CAGCCGTTTACAAAGACATGCA-3′
shmt-R: 5′-GAATGGCGTTGGTGATGGTT-3′
act42a-F: 5′-CTCCTACATATTTCCATAAAAGATCCAA-3′
act42a-R: 5′-GCCGACAATAGAAGGAAAAACTG-3′

Total RNA was extracted from NP cells using Trizol reagent (TransGen Biotech) according to the manufacturer's instructions. Total RNA (1 μg) was reverse‐transcribed into cDNA by using the Quantscript RT Kit (Beyotime) according to the manufacturer's protocol. The cDNA was then used as the template for the qPCR with TransStart® Top Green qPCR SuperMix kit (TransGen Biotech) on the Eppendorf Realplex4 instrument (Eppendorf). The reaction conditions were set as follows: 95°C for 3 min, followed by 40 cycles at 95°C for 30 s and at 55°C for 20 s, and extension for 20 s at 72°C. All primer sequences are listed in the Table 1. The 2^−ΔΔCt method was used to calculate the relative RNA expression levels of target genes.³⁴

Total RNA from intestinal tissues (in vivo) and IEC-6 cells (in vitro) was extracted. First-strand cDNA was synthesized, and target cDNA was amplified. The PCR primers were as follows: rat FoxM1 F: 5′-CAAGGTAAAAGCCACGTCTAAG C-3′, R: 5′-GGAGCAGCAGGTGACTAATGG-3′; rat Nurr1 F: 5′-CCAATCCGGC AATGACCAG-3′, R: 5′-TGATGATCTCCATAGAGCCAGTCAG-3′; rat β-actin F: 5′-CTGGAGAAGAGCTATGAGCTG-3′, R: 5′-AATCTCC TTCTGAT CCTGTC-3′; human FoxM1 F: 5′-GGAGGAAATGCCACACTTAGCG-3′, R: 5′-TAGGACTTCTTGGGTCTTGGGGTG-3′; human Nurr1 F: 5′-CATGGACCT CACCAACACTG-3′, R: 5′-AGTAAACCGACCCGGAGTG-3′; and human β-actin F: 5′-ACCCTGAAGTACCCCATCGAG-3′; R: 5′-ACATGATCTGGGTCATCTTCTCG-3′. mRNA expression was quantified using a TransStart Top Green qPCR SuperMix kit (TransGen Biotech, Beijing, China). β-Actin was used as an endogenous control, and the ΔΔCT method was used to analyze relative mRNA expression.

RT-qPCR was used to investigate the expression levels of the PI3K-AKT pathway associated genes (COL1A1, COL1A2, COL4A5, FN1, IGF2, and PIK3R1).
Total RNA extraction and cDNA synthesis were performed using the TriQuick Reagent (TRIzol Substitute) (Beijing Solarbio Science & Technology Co., Ltd.) and the UEIris II RT-PCR System for the cDNA Synthesis SuperMix (Beijing TransGen Biotech Co., Ltd.) according to the manufacturers’ instructions. All synthesized cDNA were used for the PCR amplification using the TransStart^® Top Green qPCR SuperMix kit (Beijing TransGen Biotech Co., Ltd.). The total reaction system was 20 μL. The primer sequences used were presented in Table 2 [6 (link)]. GAPDH was used as a normalization control.
The cyclic parameters used were pre-denaturation at 94 °C for 30 s, followed by the PCR reaction (45 cycles of 94 °C for 15 s, 60 °C for 15 s, and 72 °C for 10 s), while fluorescence data were collected at 72 °C. The relative gene expression levels were analyzed using the 2^−ΔΔCT method [31 (link)].

Total RNA was isolated from chick embryo liver tissues, HepG2 cells, and zebrafish by using TRIzol Reagent as described by the manufacturer’s instructions. A NanoDrop ND-2000C spectrophotometer (Thermo, Waltham, MA, USA) was used to quantify the concentration of RNA. A total of 1000 ng of the total RNA was utilized to synthesize cDNA following the manufacturer’s guide (TransGen Biotech, Beijing, China). The result of qRT-PCR was analyzed by LightCycler 96 system (Roche, Switzerland) by using the TransStart Top Green qPCR Supermix Kit (TransGen Biotech, Beijing, China). The target gene expression was assessed by using the 2 ^−ΔΔCT method and GAPDH was used as the housekeeper gene and to a control group sample. The sequences of primers were listed in Table 1.

The RNAprep Pure Plant Kit (TIANGEN, Beijing China) was used to isolate total RNA. cDNAs were obtained by total RNA reverse transcription using HiScript® II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing China). Primers for the VvHSP20 genes were designed by Primer Premier 5.0 software and listed in Additional file 2: Table S1. The grape ubiquitin1 gene was used as the reference gene [57 (link), 58 (link)] and the expression level of K1 was used as the calibrator. Quantitative real-time PCR was conducted with a total volume of 10 μL of TransStart Top Green qPCR SuperMix kit (TRANSGEN, Beijing China) in CFX96 Real-Time PCR Detection System (Bio-Rad). The relative expression changes of VvHSP20s genes were calculated using the 2^-ΔΔCt method from three independent replicates [59 (link)]. SPSS version 21.0 was employed to analyze the statistical significant differences of the gene expression levels by ANOVA with Duncan’s multiple range test.
The FPKM values of VvHSP20 genes were from the RNA-Seq data (Accession codes, SRA: PRJNA541089). The average FPKM value of each repetition was converted to log10. Pheatmap (R package) was used to generate the heatmap.