Transstart tip green qpcr supermix

The PCR reactions were performed in a 20 μl total reaction volume including 10 μl of 2 × TransStart^® Tip Green qPCR SuperMix (TransGen Biotech, China), 0.4 μl of Passive Reference Dye II, 0.4 μl of each of the gene-specific primers, 1 μl of cDNA templates, and 7.8 μl of ddH₂O. Real-time PCR was carried out with specific primers to determine the expression level of Hsp70, Hsp21, and sHsp21. β-actin was used as an internal reference gene. Reactions were carried out on the 7500 Real-Time PCR System (Applied Biosystems, Grand Island, NY, United States). The thermal cycler parameters were as follows: 94°C for 30 s followed by 40 cycles each at 94°C for 5 s, and 60°C for 34 s. At the end was the melting curve stage following the default parameters. A standard curve was derived from the serial dilutions to quantify the copy numbers of target mRNAs. The standard curves for Hsp70, Hsp21, sHsp21, and the β-actin had slopes of −3.214, −3.313, −3.325, and −3.415; correlation coefficients (R²) of 0.998, 0.994, 0.991, and 1.000; amplification efficiencies of 104.714, 100.361, 99.884, and 96.265; and Y-intercepts of 44.530, 44.201, 47.418, and 49.371, respectively. Each cDNA sample was assayed in triplicate. The data were analyzed based on the Cp method according to the mathematical model of Pfaffl (Fleige et al., 2006 (link)), simplified to 2^–△△Ct as follows:

Total RNA was extracted from the rice seedlings (ethylene-treated and untreated) and germination rice seeds (hormone-treated and untreated) with the RNAiso Plus reagent (TaKaRa, CAS No. 108-95-2). Then, total RNA was reverse-transcribed using Hifair III 1st Strand cDNA Synthesis SuperMix (Yeasen CAT: 11141ES60). The qPCR was performed using TransStart Tip Green qPCR SuperMix (TransGen Code: AQ141-04) and the MyiQ2 Real-time PCR Detection system (Bio-Rad) according to the instructions of the manufacturer. The primers used are listed in Supplementary Table 1.

Total RNA of the root, vine, and leaf samples were extracted separately using the plant RNA extraction kit (TIANGEN BIOTECH, Beijing, China) according to the manufacturer’s instructions. RNA concentration and quality were tested by NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA), with the integrity checked on 0.8% agarose gel. The expression levels of 12 CrMTPs were determined by quantitative reverse transcription PCR (qRT-PCR), and three biological replications for each treatment were conducted. In brief, a total of 1 μg of RNA was reverse transcribed into cDNA in a 20 μL reaction volume using AMV reverse transcriptase (TransGen Biotech, Beijing, China) according to the supplier’s instructions. To quantify the relative transcript levels of selected CrMTP genes, qRT-PCR was performed with gene-specific primers using the LightCycler480 system (Roche, Basel, Switzerland) and TransStart Tip Green qPCR SuperMix (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. The gene-specific primers used for this analysis are listed in Table S2. All gene expression data obtained via qRT-PCR were normalized to the expression of CrEF-1α (Table S2).

To examine the transcriptional changes of BACE1 following PGRN treatment, N2a cell lines were plated in a 12-well culture plate, and different concentrations of PGRN were added to wells. After overnight incubation, total RNAs were isolated using TRIzol^® Reagent (15596018, Invitrogen), then reversely transcribed to cDNA using the PrimeScript^® RT reagent kit (RR047A, TaKaRa). qPCR reactions were performed using TransStart^® Tip Green qPCR SuperMix (AQ141, Transgen, Beijing, China). The expression of GAPDH was used as an endogenous control. Primer sequences were as follows: BACE1 (Jiang et al., 2014 (link)), forward 5′-CAG TGG AAG GTC CGT TTG TT-3′, reverse 5′- CTA AAG GAT GCT GGG CAG AG-3′; GAPDH, forward 5′-CCC TTC ATT GAC CTC AAC TA-3′, reverse 5′-CCT TCT CCA TGG TGG TGA A-3′.
Experiments were repeated four times. Relative expression levels of BACE1 were calculated using the 2^−ΔΔCT method and normalized to GAPDH.

The qRT-PCR was performed as described previously (15 (link)). Briefly, total RNA was extracted using TransZol Up Plus RNA Kit (Transgen, Beijing, China) following the manufacturer's instructions. 0.5 μg total RNA was reverse transcribed using the EasyScript® First-Strand cDNA Synthesis SuperMix (Transgen, Beijing, China). The mRNA expression levels were measured by TransStart® Tip Green qPCR SuperMix (Transgen, Beijing, China) on a Roche 480 real-time PCR system thermocycler. Each sample was analyzed in triplicates and target gene expression was analyzed by 2^−ΔΔCt method (16 (link)), following normalization with β-actin gene. The primers used are provided in Table 1.

For validation of the miRNA deep sequencing results, 10 DEMs (five up- and five down-regulated) were randomly selected, and a portion of the RNA preparations used in miRNA deep sequencing was used for qRT-PCR analysis. Total RNA was treated by RNase-free DNase (Qiagen, Germany) to remove any residual DNA contamination. Reverse transcription of total RNA was performed using TransScript Green miRNA Two-Step qRT-PCR SuperMix (TransGenBiotech, China) according to the recommendations in the product manual. We used TransStart TipGreen qPCR SuperMix (TransGen Biotech, China) to conduct qRT-PCR analysis on an ABI 7300 Real-time PCR system (Applied Biosystems, USA) following reaction conditions suggested by TransGenBiotech. All reactions were analyzed in triplicate. The relative quantification of each miRNA was calculated based on the 2^-ΔΔCT method [53 (link)]. All primers are listed in Supplementary Table 6. A U6 gene was selected as the internal control. IBM SPSS Statistics 22 was employed for statistical analysis.