Chamq universal sybr qpcr master mix kit

Real-time quantitative PCR (RT-qPCR) was performed using the cDNA samples returned by the sequencing company. The RT-qPCR primers were designed using Primer Premier v6.0 (http://www.premierbiosoft.com/primerdesign/index.html). The Actin 4 gene of maize was selected as the internal reference gene. The RT-qPCR program uses a Light Cycler 480 system (Roche, Roche Diagnostics, Basel, Switzerland) and a 2× ChamQ Universal SYBR qPCR Master Mix Kit (Vazyme, Q711, Vazyme biotech, Nanjing, China). Each RT-qPCR was biologically repeated three times. The relative expression of the candidate genes was calculated using the following formula:

Differentially expressed candidate genes were selected based on the results of gene annotation for further verification of their expression in extreme materials. By comparing the annotations of functions of the differentially expressed genes in the database, we found that 3 key genes were involved in hormone regulation or regeneration and speculated that these genes might be involved in somatic embryogenesis.
Real-time quantitative PCR (RT-qPCR) primers were designed using Primer Premier v6.0 (http://www.premierbiosoft.com/primerdesign/index.html). The actin 4 gene of soybean was selected as the internal reference gene. The material RNA was extracted by TRIzol, and the genomic DNA was then removed by a 4× gDNA wiper mix (Vazyme, R223, Vazyme biotech, Nanjing, China). The RNA was retrieved as a single-strand cDNA by a 5× HiScript II qRT SuperMix II (Vazyme, R223, Vazyme biotech, Nanjing, China). The RT-qPCR program used a Light Cycler 480 system (Roche, Roche Diagnostics, Basel, Switzerland) and the 2× ChamQ Universal SYBR qPCR Master Mix Kit (Vazyme, Q711, Vazyme biotech, Nanjing, China). The RT-qPCR analysis was carried out based on 3 biological replicates. The relative expression of the candidate genes was calculated according to the following formula:

Absolute quantitative PCR was performed to analyze the integrated copy number of the deletion cassette in disruption mutant according to the reported method [31 (link),32 (link),33 (link)]. The copy number of the G418 resistance gene (neo) in creA disruption cassette in the creA disruption mutant was determined. The standard curve was measured using a plasmid (M13-PTgpd-neo) which contains single neo gene as the template.
The relative transcriptional level of cellulytic genes in H. insolens was determined by qRT-PCR. β-actin gene was used as an internal control for normalization. Reactions were performed using 2×ChamQ Universal SYBR qPCR Master Mix kit (Vazyme, Nanjing, China) as described by the manufacturer’s protocol. Data analysis was done with the 2^−△△Ct method.
The qPCR experiment was performed on the LightCycler 96 System as follows: 1 cycle of 94 °C for 5 min, followed by 40 cycles of 94 °C for 5 s, 58 °C for 15 s, and 72 °C for 20 s. The primers for qPCR are shown in Table S1. Three biological replicates were performed with three technical replicates for each biological replicate.

Different tissues (cotyledon, hypocotyl, and radicle) of WR were selected on the fifth day during the sprout stage, and hypocotyls of the four samples (WR, WN, SR, and SN) were selected on the fourth, fifth and sixth days. Each sample had three biological repeats. Total RNA was extracted using RNA isolater Total RNA Extraction Reagent (Vazyme Biotech, China). After the extraction, the RNA samples were electrophoresed in 1% agarose gel for detection and quality control. Additionally, a NanoDrop spectrophotometer was used to check the concentration and purity of the RNAs. Samples of poor quality were discarded. Premier 5.0 (Pi et al., 2018 (link)) was used to design the RT-qPCR primers (Supplementary Table 2). PvC3Hs were selected from the GO enrichment and KEGG pathways, and some PvC3Hs were selected as candidate genes for verification. ACTIN-11 of common bean served as an internal control gene (Borges et al., 2012 (link)). The RNA was transcribed to cDNA, and then, RT-qPCR was performed using 2 × ChamQ Universal SYBR qPCR Master Mix Kit (Vazyme Biotech, China). The RT-qPCR was performed in triplicate. The relative gene expression was determined as follows (Livak and Schmittgen, 2001 (link)):
Relative Expression = 2^ΔΔCt, Where ^ΔΔCt = [Ct(Pv target genes) −Ct(PvACTIN-11)].

To conduct ChIP studies, 2.5 × 10⁵ HNE1 or C666-1 cells were treated with 10 μM P8 fibrils or 10 μM P8 fibrils plus 50 nM 17-AAG for 24 h. ChIP was performed by following the manufacturer’s instructions for the SimpleChIP® Enzymatic Chromatin IP Kit (#9003, Cell Signaling Technology, Danvers, USA). qPCR was performed by using a ChamQ Universal SYBR qPCR Master Mix Kit (#Q711, Vazyme, Nanjing, China) on a LightCycler® 480 (Roche, Basel, Switzerland). The primers used are listed in Supplementary Table 2. qPCR was performed in triplicate for all ChIP experiments, and error bars depicting standard deviations are shown for all values. The input values were calculated using the following equation: ΔCT = CT(ChIP)-[CT(Input)-LogE(Input dilution factor)], % Input=E-ΔCT (E: specific primer efficiency value).

Cells were cultured and treated with 10 μM P8 fibrils, followed by treatment with an additional 50 nM 17-AAG or STA9090. Untreated cells were used as corresponding controls. RNA was isolated from cells by TRIzol (Invitrogen) following the manufacturer’s instructions. The RNA concentrations were determined by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). The HiScript® III RT SuperMix for qPCR Kit (Q225, Vazyme, Nanjing, China) was used for reverse transcription. Then, the relative mRNA levels were detected using the ChamQ Universal SYBR qPCR Master Mix Kit (#Q711, Vazyme, Nanjing, China). The primers used are listed in Supplementary Table 2.

Gene expressions of C3 were determined by qRT-PCR. Total RNA was isolated from the lung tissues of mouse and human COPD patients, and the cultured cells were treated with Trizol reagent (Thermo Fisher Scientific, MA, USA) and the Nano Drop 2000 was used to measure RNA concentration. RNA was then reverse transcribed into cDNA using the HiScript III RT Supermix for qPCR (+gDNA wiper) Kit (R323-01, Vazyme, Nanjing, China). The qPCR reactions were performed on the Applied Biosystems^® QuantStudio^® 5 in a 20 μl reaction system using the ChamQ Universal SYBR qPCR Master Mix Kit (Q711-02, Vazyme, Nanjing, China).