Total RNA is extracted from cells by Trizol Company and OD value is measured to quantify RNA concentration. Olig (dT) 15 is used as a primer and RNA reverse transcription kit (TOYOBO Company) is used to prepare the DNA. The DNA was used as a template. The total reaction system is 20 μL according to the operation of Realtime PCR Master Mix instructions. The reaction conditions are set as follows: after pre-denaturation for 30 s at 95 °C, denaturation for 5 s at 95 °C, annealing for 5 s at 56 °C, and elongation for 15 s at 72 °C. According to Livak and Schmittgen's comparison threshold method, the CT value is read. The expression of VCAM-1 is calculated by equation 2−△△Ct.
Rna reverse transcription kit
Manufactured by Toyobo
Sourced in China, Japan
The RNA reverse transcription kit is a laboratory tool used to convert RNA molecules into complementary DNA (cDNA) through the process of reverse transcription. This kit provides the necessary enzymes, buffers, and reagents required to perform this fundamental step in many molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Sybr premix ex taq 2, by Novoprotein (3 mentions)
Cfx manager, by Bio-Rad (2 mentions)
Kod sybr qpcr mix kit, by Toyobo (1 mentions)
E z n a total rna kit, by Omega Bio-Tek (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Intracellular fixation and permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Dna marker, by Sangon (1 mentions)
Trizol, by Merck Group (1 mentions)
Rna extraction kit, by Tiangen Biotech (1 mentions)
8 protocols using rna reverse transcription kit
1
Real-time PCR for VCAM-1 Expression
2
Quantitative Analysis of GATA3 and CHI3L1 Gene Expression
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The expressions of GATA3 and CHI3L1 genes mRNA were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). Briefly, the HUVECs’ total RNA was extracted using E.Z.N.A. Total RNA Kit (OMEGA Bio-Tek, USA), respectively, and the aforementioned samples total RNA was reversely transcribed into cDNA using the RNA reverse transcription kit (TOYOBO, Japan). RT-PCR was then carried out by KOD SYBR qPCR Mix kit (TOYOBO, Japan) and targeting primers. The reaction system was utilized for 20 uL, with reaction conditions of 98°C (2 minutes), 40 cycles of 98°C (10 seconds), 60°C (10 seconds) and 68°C (30 seconds). The melting curve was then analyzed. Experimental apparatus used LightCycler 480 II real-time fluorescent qPCR. The qRT-PCR data were quantified using 2−ΔΔCt. All gene primer sequences are shown as follows:
3
Quantitative Transcriptome Analysis of Fruit Pulp
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Total RNA was extracted from fruit pulp tissue at different developmental stages of the three cultivars using RNAprep Pure (Tiangen, Beijing, China). First-strand cDNA was synthesized using an RNA reverse transcription kit (Toyobo, Shanghai, China). To verify the accuracy of the transcriptome data, we used Primer 5 to design specific primers for 12 genes and performed qRT–PCR analysis (Table S1 ). These primers were synthesized by Sangon Biotech (Sangon, Shanghai, China). The 2−ΔΔCT method was used for relative quantitative analysis of the data, and the internal reference gene TATA box binding protein-like (TBP) [35 (link)] and SYBR Premix Ex Taq II (Novoprotein, Shanghai, China) were employed to validate the DEG expression results. Three biological and three technical replicates of each reaction were analyzed on a LightCycler® 480 instrument (Roche, Switzerland) with a first step of 95 °C for 5 min for pre-denaturation, followed by 40 cycles of 95 °C for 10 s for denaturation, and 60 °C for 30 s for annealing/extension [35 (link)]. All primers are listed in Table S1 .
4
Muscarinic Receptor Modulation Study
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Reagents involved in this study include: RNA reverse transcription kit (TOYOBO company), TaqMan real-time fluorescence quantitative kit (CWBiao, Suzhou, China), CNO (Selleck Corporation, Shanghai, China), Milameline hydrochloride (MH, Selleck Corporation, Shanghai, China), VU0238429 (VU, Selleck Corporation), anti-mAChR4 antibody (18C7.2, Abcam, UK), and Intracellular Fixation and Permeabilization Buffer (eBioscience, Thermo Fisher Scientific, US).
5
Molecular Profiling of Inflammatory Signaling
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Trizol (Sigma Company, USA); RNA reverse transcription kit (TOYOBO), SYBY® κGreen Real-time PCR Master Mi× (TOYOBO), and 5×Loading Buffer (TAkara Company, USA); DNA Marker (Shanghai Sangon Biotech Co., Ltd.); paraffin, paraformaldehyde, and hematoxylin-eosin (HE) staining reagent (Shanghai Beyotime Institute of Biotechnology), protein extraction kit and sodium-dodecyl-sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE) kit ((Shanghai Beyotime Institute of Biotechnology); electrochemiluminescence (ECL) liquid (Applygen Technology Company); goat anti-rabbit immunoglobulin G (IgG)/horseradish peroxidase (HRP), goat anti-rabbit MyD88, TLR 4, and NF-κB (ab102890, ab16894, ab22048, and ab22146, Abcom Company, USA); TNF-α, IL-6, and IL-β enzyme-linked immuno-sorbent assay (ELISA) kits (Shanghai Beyotime Institute of Biotechnology). Primers were designed by the Shanghai Sangon Biotech Co., Ltd.
6
Validating Transcriptome Profiles via qRT-PCR
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RNAprep Pure (Tiangen, Beijing, China) was used to extract total RNA from the scion leaf tissue of HAM, HDM and HM. The RNA reverse-transcription kit (Toyobo, Shanghai, China) was used to form the first-strand cDNA. Primer 5 was used to create unique primers for 12 randomly selected genes, and qRT-PCR analysis was performed to validate the transcriptome results (Table S11 ). These primers were synthesized by Tsingke Biotech (Beijing, China). To confirm the differential gene expression patterns, qRT-PCR was carried out using Bio-Rad CFX Manager (Bio-Rad, Shanghai, China) and SYBR Premix Ex Taq II (novoprotein, Shanghai, China), with all samples having undergone three biological and technical replications. The 2−∆∆CT method [75 (link)] was used to calculate gene expression levels after normalizing them against the geometric mean of the citrus reference gene, Actin (GenBank: XM_006429010.2).
7
Transcriptome Analysis of Citrus Fruit Development
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Total RNA was extracted from fruit pulp tissue at different developmental stages of the three cultivars using RNAprep Pure (Tiangen, Beijing, China). The first-strand cDNA was synthesized using an RNA reverse transcription kit (Toyobo, Shanghai, China). To verify the accuracy of the transcriptome data, we used Primer 5 to design specific primers for 12 genes and performed qRT-PCR analysis (Table S5 ). These primers were synthesized by Tsingke Biotech, Beijing, China. The qRT-PCR analysis was performed using Bio-Rad CFX Manager (Bio-Rad, Shanghai, China) and SYBR Premix Ex Taq II (novoprotein, Shanghai, China) to validate the DEG expression results. All samples had three biological replicates and were technically replicated three times. Gene expression levels were normalized against the geometric mean of the citrus reference gene β-actin (GenBank: XM_006429010.2) and calculated by the 2−ΔΔCT method [31 (link)].
8
Quantification of HIF-1α Expression
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The cells of the blank control group and CoCl2 group were extracted with an RNA extraction kit (TIANGEN Company, Beijing, China) and reversely transcribed into cDNA using an RNA reverse transcription kit (TOYOBO, Japan). qPCR was carried out according to the instructions of TOYOBO Company. The reaction volume was 20 μL, including 10 μL Master Mix, 1 μL cDNA, 1 μL Forward Primer, 1 μL Reverse Primer, and 7 μL deionized water. Then, a relative quantitative (RQ) value (RQ = 2-ΔΔCT) was calculated, which represents the relative expression level of the genes. The following primer sequences were used:
GAPDH: (5’-3’) Forward primer GGAAGGACTCATGAC CACAGT
Reverse primer GGCAGGTTTTTCTAGAC GGC
HIF-1α: (5’-3’) Forward primer GGCAGCAACGACA CAGAAAC
Reverse primer TGCAGGGTCAGCAC TACTTC
GAPDH: (5’-3’) Forward primer GGAAGGACTCATGAC CACAGT
Reverse primer GGCAGGTTTTTCTAGAC GGC
HIF-1α: (5’-3’) Forward primer GGCAGCAACGACA CAGAAAC
Reverse primer TGCAGGGTCAGCAC TACTTC
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