The RNA prep Pure Plant Plus Kit (TaKaRa, Dalian, China) was used to extract the RNA from berry, root, leaf, stem and inflorescence from different V. vinifera varieties under different treatments. A BIO-RADXR gel imaging analysis system (Bio-Rad, CA, USA) was established to analyze the purity and integrality of RNA. According to the manufacturer’s instructions, we used the PrimeScript™ RT reagent kit (TaKaRa, Dalian, China) with 1 μg of total RNA to perform qRT-PCR analysis. The qPCR system employed the following program: 95 °C for 20 s, then followed by 39 cycles of 95 °C for 15 s, finally, 55 °C for 15 s and 60 °C for 15 s. The 2−ΔΔCt assay was used to calculate the final relative expressions of each gene. The primers for qPCR analysis are all listed in Table S1 .
Bio radxr gel imaging analysis system
Manufactured by Bio-Rad
Sourced in United States, China
The BIO-RADXR gel imaging analysis system is a versatile and powerful tool for capturing and analyzing images of gels, blots, and other samples. It features high-sensitivity CCD camera technology, advanced imaging software, and a user-friendly interface to provide accurate and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Rna prep pure plant plus kit, by Takara Bio (4 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Gdna eraser perfect real time, by Takara Bio (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca assay, by Beyotime (1 mentions)
Ab172570, by Abcam (1 mentions)
Ab214430, by Abcam (1 mentions)
Ab221679, by Abcam (1 mentions)
Ab2324, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Sds page electrophoresis system, by Bio-Rad (1 mentions)
Blocking buffer, by Beyotime (1 mentions)
Bca kit, by Abcam (1 mentions)
Ab202985, by Abcam (1 mentions)
Ab96899, by Abcam (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Ab223352, by Abcam (1 mentions)
6 protocols using bio radxr gel imaging analysis system
1
Comprehensive RNA Extraction and qRT-PCR Analysis
2
Grape Root RNA Extraction and qRT-PCR
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The extraction of total RNA in the grape roots of the different treatments was carried out on an RNA Prep Pure Plant Plus Kit (TaKaRa, Dalian, China). A BIO-RADXR gel imaging analysis system (Bio-Rad, CA, United States) was used to determine the purity and integrity of the RNA extracted. One microgram of total RNA was extracted by the use of a PrimeScript™ RT Reagent Kit in combination with gDNA Eraser (Perfect Real Time) (TaKaRa, Dalian, China) according to the manufacturers’ instructions. Then, first-strand cDNA was obtained.
The total final volume was 10 μl, consisting of 1 μl of cDNA, 5 μl of TB Green® Fast qPCR Mix, 3 μl of ddH2O, and 1 μl of a forward and reverse primer mixture. Then, qRT-PCR was implemented in a CFX connect Real Time PCR Detection System (Bio-Rad, CA, United States), and the sequences of all the genes and transcription factors identified in this study were acquired from the EnsemblPlants database2 . The qPrimerDB-qPCR Primer Database3 was used to design the primers used in this study. The specific information of all the primer sequences used is shown in Supplementary Table 1 , and the reactions were carried out as follows: 95°C for 20 s, followed by 39 cycles of 95°C for 15 s, 55°C for 15 s and 60°C for 15 s. The 2–ΔΔCt method was used for the determination of relative gene expression.
The total final volume was 10 μl, consisting of 1 μl of cDNA, 5 μl of TB Green® Fast qPCR Mix, 3 μl of ddH2O, and 1 μl of a forward and reverse primer mixture. Then, qRT-PCR was implemented in a CFX connect Real Time PCR Detection System (Bio-Rad, CA, United States), and the sequences of all the genes and transcription factors identified in this study were acquired from the EnsemblPlants database
3
Protein Expression Analysis of SW982 Cells
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Total protein was extracted from the SW982 cells or tumor tissues using RIPA lysis buffer (Beyotime, China), followed by separation using an SDS-PAGE electrophoresis system (Bio-Rad, USA) after the total protein was measured by performing bicinchoninic acid (BCA) assay (Beyotime, China). Then, the protein samples were transferred from the gel onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Germany) and blocked with 5% skimmed milk for 12 h at 4°C, followed by incubation with the HOXA13 antibody (ab172570, 1 : 1000, Abcam, Cambridge, UK), VEGFR2 antibody (ab221679, 1 : 1000, Abcam, Cambridge, UK), Ki67 (ab16667, 1 : 1000, Abcam, Cambridge, UK), cleaved caspase-3 antibody (ab214430, 1 : 5000, Abcam, Cambridge, UK), and cleaved caspase-9 antibody (ab2324, 1 : 1000, Abcam, Cambridge, UK) for 12 h at 4°C. The protein blots were visualized using the ECL kit (Thermo Scientific, China) and the Bio-Rad XR gel imaging analysis system (Bio-Rad, USA) after incubation with Goat Anti-Rabbit IgG H&L antibody (1 : 10000). All antibodies were purchased from Abcam (UK).
4
Western Blot Analysis of TNF-α Signaling
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Protein extracted from mice by RIPA lysis buffer (Solarbio, China) was measured by BCA kit (Abcam, UK) to determine the protein concentration, followed by the separation of SDS-PAGE system (Bio-Rad, USA) and subsequently transferred to PVDF membrane (Millipore, Germany). Then, membrane was blocked by blocking buffer (Beyotime, China) at 4°C for 4 h, followed by the incubation with the primary antibodies of TNFα (ab183218, 1:1000), TNFR1 (Abcam, ab223352, 1:1000), TNFRSF1A Associated Via Death Domain (TRADD) (Abcam, ab110644, 1:1000), RIP (Abcam, ab202985, 1:1000), TNF Receptor Associated Factor 2 (TRAF2) (Abcam, ab244317, 0.4 μg/mL), p-Inhibitor of Nuclear Factor Kappa B Kinase Subunit Beta (p-IKKβ) (Cell Signaling, #2697, 1:1000), IKKβ (Cell Signaling, #8943, 1:1000), p-p65 (Cell Signaling, #3033, 1:1000), p65 (Cell Signaling, #8242, 1:1000), PAR2 (Abcam, ab180953, 1:10,000), PKC-γ (Abcam, ab71558, 1: 2000), PKA (Cell Signaling, #4782, 1:1000) and TRPV1 (Abcam, ab6166, 1:1000) for 12 h at 4°C. Following the incubation with the secondary antibody linked to HRP (Abcam, ab96899, 1:10,000) for 4 h at room temperature, protein was measured by ECL kit (Thermo Scientific, China) and Bio-Rad XR gel imaging analysis system (Bio-Rad, USA).
5
Grape Berry RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from grape berries using the RNA Prep Pure Plant Plus Kit (TaKaRa, Dalian, China). BIO-RADXR gel imaging analysis system (Bio-Rad, CA, USA) was used to detect the purity and integrality of RNA extracted. qRT-PCR was performed using the CFX Connect Real-Time PCR Detection System (Bio-Rad, CA, USA). The steps were as follows: 95°C for 20 s, followed by 95°C for 39 cycles (15 s), 55°C for 15 s, and finally, 60°C for 15 s. The 2-△△Ct assay determined the genes’ relative expression. All genes and transcription factor sequences were acquired from EnsemblPlants (http://plants.ensembl.org/info/about/index.html ). Furthermore, the primers for qRT-PCR were designed qPrimerDB-qPCR Primer Database (https://biodb.swu.edu.cn/qprimerdb/ ). All specific information of all sequences is presented in Table 1
6
Comprehensive Grapevine Transcriptome Analysis
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RNA extraction was carried out on RNA prep Pure Plant Plus Kit (TaKaRa, Dalian, China), which used root, stem, leaf, flower, berry, bud from grapevine in all sampling stages, Purity and completeness were analyzed using the Bio-RadXR Gel Imaging analysis system (Bio-RAD, CA, USA). Following the manufacturer's instructions, the PrimeScript™RT Kit with gDNA Eraser (Perfect Real time) (TaKaRa, Dalian, China) used 1 μg of total RNA extracted from collected samples, designed to obtain the first strand of cDNA.
The final composition volume was 10 μL, consisting of 1 μL cDNA, 5 μ L TB Green®Fast qPCR Mix, 3 μL ddH2O and 1 μL positive and negative primer mixture. qRT-PCR was then performed using CFX coupled with Real Time PCR detection system (Bio-RAD, CA, USA). All sequences of genes and transcription factors using in this study were downloaded from EnsemblPlants (http://plants.ensembl.org/info/about/index.html ). The specific primers (55 genes, 7 transcription factors and actin) were designed using qPrimerDB (https://biodb.swu.edu.cn/qprimerdb/browse_plants ), and the sequences and information of genes and transcription factors were all listed in Table S1 . qRT-PCR procedure :95 °C was treated for 20 s, followed by 95 °C for 15 s, 55 °C for 15 s, and 60 °C for 15 s, with 39 cycles. The final relative expression of each gene was analyzed by 2−△△Ct method.
The final composition volume was 10 μL, consisting of 1 μL cDNA, 5 μ L TB Green®Fast qPCR Mix, 3 μL ddH2O and 1 μL positive and negative primer mixture. qRT-PCR was then performed using CFX coupled with Real Time PCR detection system (Bio-RAD, CA, USA). All sequences of genes and transcription factors using in this study were downloaded from EnsemblPlants (
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