To determine the relative expression of the empty vector, Wt- and I21N-CRYBB2 in transfected cells, RT-qPCR was performed. Total RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), qPCR SYBR® Green Master Mix (Vazyme Biotech, Co., Ltd.) and the RNA was reverse transcribed into cDNA using a QuantiTect Reverse Transcription kit (cat. no. 205313, Qiagen China Co., Ltd.), according to the manufacturer's protocol. qPCR was performed using a PCR amplifier (26 (link)). The method of gene quantification used was 2-ΔΔCq as previously described (27 (link)). PCR primers were designed by Invitrogen (Thermo Fisher Scientific, Inc.) as follows: CRYBB2 forward, 5'-GTAGCCAGGATTCTGCCATAGGAA-3' and reverse, 5'-GTGCCCTCTGGAGCATTTCATAGT-3'; GAPDH forward, 5'-TTCCGAGTTCCTGTCCCTAATG-3' and reverse, 5'-GCCTCCTTCACCTTCTGCTTG-3'. qPCR was performed using the FTC2000 (Funglyn Biotech). Samples were set up in 50 µl final volumes containing 6 µl 5X PCR buffer, 0.6 µl 2X primers (25 pmol/µl), 0.3 µl probe (25 pmol/µl) or 0.3 µl SYBR-Green, 1 µl dNTPs (10 mM), 0.3 µl Taq enzyme (5 U/µl), 3 µl Mg2+ (25 mM), 1 µl template and 17.2 µl DEPC water (Sigma-Aldrich; Merck KGaA). Thermocycling conditions were as follows: Initial denaturation at 94˚C, followed by 40 cycles of 20 sec at 94˚C and 30 sec at 60˚C. The relative expression was calculated based on the expression of GAPDH.
Quantitect reverse transcription kit
Manufactured by Vazyme
Sourced in China
The QuantiTect Reverse Transcription kit is a reagent system designed for cDNA synthesis from RNA samples. It enables the conversion of RNA into complementary DNA (cDNA) for use in downstream applications such as real-time PCR.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Plant rna kit, by Tiangen Biotech (2 mentions)
Depc water, by Merck Group (1 mentions)
Sybr green qpcr master mix, by Vazyme (1 mentions)
Aceq universal sybr qpcr master mix, by Vazyme (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Recombinant human il 1β, by Thermo Fisher Scientific (1 mentions)
Adamts5, by Abcam (1 mentions)
Type 2 collagen, by Abcam (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Sybr green master mix, by Takara Bio (1 mentions)
Cfx96 real time pcr instrument, by Bio-Rad (1 mentions)
Sybr premix ex taq, by Vazyme (1 mentions)
Abi 7500 system, by Thermo Fisher Scientific (1 mentions)
Miseq benchtop sequencer, by Illumina (1 mentions)
7 protocols using quantitect reverse transcription kit
1
Quantifying CRYBB2 Isoform Expression
2
Hippocampus Total RNA Extraction and Analysis
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The total RNA extraction was performed according to the manufacturer’s instructions. Briefly, the hippocampus was homogenized with 1 mL TRIzol reagent (15596026, Invitrogen, Carlsbad, CA, USA) and incubated for 10 min at room temperature (RT). Chloroform (200 µL) was added and mixed by vigorous shaking for 30 s and standing for 10 min at RT. The RNA precipitation was obtained after centrifugation (12,000 rpm for 15 min) and washed once with 75% ethanol. The precipitates were finally dissolved in 20 µL diethylpyrocarbonate (DEPC) water. A measure of 1 µg of total RNA was used to perform reverse transcription by the QuantiTect Reverse Transcription Kit (Vazyme, Nanjing, China), and real-time PCR was performed with AceQ Universal SYBR qPCR Master Mix (Vazyme, China) on an ABI7500. The amplification reaction condition was set as follows: denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 30 s. The expression of fold changes for target genes was calculated by the 2−ΔΔct method. The primer sequences were listed in Table 1 .
3
Morusin Modulates Inflammatory Pathways
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Morusin was obtained from MCE (New Jersey, USA), dissolved in dimethylsulfoxide (DMSO), and diluted in cell culture medium so that DMSO < 0.1% of the total volume. Recombinant human IL-1β, obtained from Peprotech (New Jersey, USA), was dissolved in water, and diluted in cell culture medium to a concentration of 10 ng/m for use in the study. Dulbecco’s Modified Eagle Medium (DMEM/F12), fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco (Rockville, MD, USA). Primary antibodies against actin, Type II Collagen and ADAMTS5 were purchased from Abcam (Cambridge, MA, USA), iNOS, COX-2, aggrecan, MMP-3, MMP-13, p65, P-p65, IκBα, P-erk, erk, P-JNK, JNK, P-p38, p38, PI3K, P-AKT, AKT were purchased from CST (Cambridge, MA, USA). RNAiso plus and SYBR Green Master Mix were purchased from Takara (Japan); and QuantiTect Reverse Transcription kit was purchased from Vazyme (Nanjing, China). All other reagents were purchased from Sigma-Aldrich (St Louis, MO, USA) unless otherwise stated.
4
Quantitative gene expression analysis
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Total RNA was extracted from young panicles using the Plant RNA Kit (Tiangen, Beijing, China) according to the manufacturer's instructions. Removal of contaminating genomic DNA and cDNA synthesis were performed using the QuantiTect Reverse Transcription kit (Vazyme, Nanjing, China). The primers were designed using QuantPrime (http://www.quantprime.de/). qRT-PCR reactions were performed as previously described (Zhou et al. 2018). The rice ATP6 and Actin (Os03g0718100) genes were used as internal controls for normalization of gene expression. Quantitative real-time PCR (qRT-PCR) assays for WA352 and candidate genes were performed with two biological replicates using SYBR Premix Ex Taq (Vazyme, Nanjing, China) on a CFX96 Real-Time PCR instrument (Bio-Rad, Hercules, CA, USA). Relative gene expression levels were calculated by the 2 ΔΔCt method (Livak and Schmittgen 2001). The names and sequences of the gene-speci c primers are given in Supplemental Table 1.
5
Transcriptional Profiling of Rice Immune Response
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Total RNA was extracted with an RNA Prep Pure Plant kit (Zomanbio, Beijing, China) following the manufacturer’s instructions. Each RNA sample (1 μg) was reverse-transcribed using the QuantiTect reverse transcription kit (Vazyme, Nanjing, China). The real-time quantitative PCR (RT-qPCR) was performed using an ABI 7500 system (Thermo Fisher Scientific, Waltham, MA, USA) with the SYBR Premix Ex Taq (Genestar, Beijing, China). Leaves of five-leaf-stage WT and mld1 plants were used to detect the expression of OsMLD1 and several defense marker genes. Two-leaf-stage WT seedling leaves, stems, and roots, as well as the adult flag leaves (1 L), top three leaves (2–4 L), young panicles, and stems, were used for tissue-specific expression analysis of OsMLD1, respectively. All of the experiments were performed with three technical replicates and three biological replicates. The OsACTIN gene in rice was used as an internal control. The sequences of all of the primers used in the study are listed in Table S7 . Additionally, the primer information of some genes, including senescence-associated genes and PR genes, was obtained from previous studies [19 (link),22 (link)].
6
Methylation Analysis of CAMTA1 Promoter
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The methylation level of the promoter region of the CAMTA1 gene in 100 IS cases vs. 100 healthy control samples was detected by methyl target region methylation sequencing. MethylTarget™ assays (targeted bisulfite sequencing) developed by Genesky Biotech (Shanghai, China) were carried out as previously described. Briefly, CpG sites adjacent to the promoter region of the CAMTA1 gene were analyzed, and based on these CpG sites, four CpG regions from CpG sites in CAMTA1 were sequenced (the relative distance from the transcriptional start site, amplification primers, and product size of these CpG regions are described in Tables 2 , 3 ). Genomic DNA was converted with bisulfite, and PCR was performed to amplify the targeted DNA sequences. The products were sequenced by an Illumina MiSeq benchtop sequencer (Illumina, CA, United States).
The total RNA was extracted using the Tiangen reagent (Beijing, China, Catalog Number: DP424). Using a QuantiTect Reverse Transcription kit (Vazyme, Wuhan, China, Catalog Number: R333-01), 2 μg of each RNA was reverse transcribed into cDNA. Expression levels of the genes were analyzed using a QuantiTect SYBR Green PCR kit (Vazyme, Nanjing, China, Catalog Number: Q221-01). The primer sequences are listed below:
The total RNA was extracted using the Tiangen reagent (Beijing, China, Catalog Number: DP424). Using a QuantiTect Reverse Transcription kit (Vazyme, Wuhan, China, Catalog Number: R333-01), 2 μg of each RNA was reverse transcribed into cDNA. Expression levels of the genes were analyzed using a QuantiTect SYBR Green PCR kit (Vazyme, Nanjing, China, Catalog Number: Q221-01). The primer sequences are listed below:
7
RNA Extraction and cDNA Synthesis
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Total RNA was extracted from young panicles using the Plant RNA Kit (Tiangen, Beijing, China) according to the manufacturer's instructions. Removal of contaminating genomic DNA and cDNA synthesis were performed using the QuantiTect Reverse Transcription kit (Vazyme, Nanjing, China). The primers were designed using QuantPrime (http://www.quantprime.de/
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