Total RNA extraction kits (Tiangen Biotech Co., Ltd., Beijing, China) were used to extract RNA. Agarose gel electrophoresis was used to evaluate RNA quality, and UV spectrophotometry was used to detect the concentration of RNA. We used 1 µg of total RNA to synthesize the first strand cDNA according to the instructions of the transcript reverse transcription kit (Tiangen Biotech Co., Ltd., Beijing, China), and we froze the product at –80°C for use in the next experiment.
Total rna extraction kit
Manufactured by Tiangen Biotech
Sourced in China
The Total RNA Extraction Kit is a laboratory tool designed to efficiently extract and purify total RNA from a variety of sample types, including cells, tissues, and microorganisms. The kit utilizes a specialized lysis buffer and silica-based columns to capture and isolate the total RNA content, providing a reliable and reproducible method for RNA extraction.
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Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (13 mentions)
Primescript rt reagent kit, by Takara Bio (8 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (6 mentions)
Sybr green pcr master mix, by Tiangen Biotech (5 mentions)
Fast quant rt super mix reverse transcription kit, by Transgene (5 mentions)
Cdna synthesis kit, by Takara Bio (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Lightcycler 96, by Roche (3 mentions)
Sybr premix ex taq, by Takara Bio (3 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (3 mentions)
5xall ln one rt mastermix kit, by Applied Biological Materials (3 mentions)
Sybr premix ex taq 2, by Takara Bio (3 mentions)
Reverse transcription kit, by Takara Bio (3 mentions)
Abi 7500 fast real time pcr system, by Thermo Fisher Scientific (3 mentions)
Sybr green real time rt pcr, by Takara Bio (3 mentions)
Cfx96 real time pcr system, by Bio-Rad (3 mentions)
Taq pcr mastermix, by Tiangen Biotech (3 mentions)
Reverse transcription kit, by Vazyme (2 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (2 mentions)
141 protocols using total rna extraction kit
1
Total RNA Extraction and Reverse Transcription
2
Royal Gala Seedling Root Total RNA Extraction and qRT-PCR
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Total RNA was extracted from “royal gala” seedlings roots via polysaccharide rich polyphenols total RNA extraction kits (TianGen, China). First-strand cDNA was obtained by Prime ScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Japan). qRT-PCR was performed three independent biological replicates on LightCycler® 96 (Roche) using the TB Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa, Japan) according to the specification. All primer sequences for qRT-PCR were given in Supplementary Table 1 , and Md-actin was used as the internal control gene. The data were analyzed by 2–ΔΔCt method (Livak and Schmittgen, 2001 (link)).
3
Investigating LINC00922 Regulation of EMT
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Fetal bovine serum was purchased from Serana (Germany), and RPMI-1640 medium was purchased from Hyclone (Beijing, China). Puromycin and polybrene were purchased from Beyotime (Shanghai, China). Total RNA extraction kits were purchased from Tiangen (Beijing, China). Reverse transcription kits and quantitative real-time PCR kits were purchased from Vazyme (Nanjing, China). LINC00922 siRNA lentivirus (si-LINC00922) and negative control (NC) lentivirus (viral titer: 8 × 108TU/ml) were purchased from GenePharma (Shanghai, China). The sequences of siRNA were shown in Supplementary Table S2 . The 24 well plates and the matrigel (356231) for transwell assay were purchased from Corning Incorporated (USA). The antibodies include GAPDH (60004-1-Ig), E-Cadherin (20874-1-AP), N-Cadherin (22018-1-AP), and vimentin (10366-1-AP) antibodies were purchased from Proteintech (Hubei, China).
4
Expression of Immune Genes in Shrimp
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Total RNA was extracted from the gill tissues of shrimp within similar sizes and injected 25 μL inactivated V. alginolyticus (1.25 × 107 CFU/mL) from each treatment according to the manufacturer's instructions for Total RNA Extraction Kits (Tiangen, China). Isolated RNA quantity and quality were determined using a NanoDrop 2000 (Thermo, US) spectrophotometer and a 1% agarose gel electrophoresis. The extracted total RNA was transferred to cDNA by PrimeScript™ RT reagent Kits (Takara, Japan). Gene-specific primers were designed using Primer Premier 5.0 software based on the cDNA sequences in GenBank (Toll-like receptor 2 (GenBank: DQ923424.1) and immune deficiency (IMD) (GenBank: FJ592176.1)) (Table 2 ). The procedure of amplification and quantitative PCR were performed per the method of Yan et al. [16 (link)]. The relative expression levels of target genes were calculated using the 2-ΔΔCt method described by [18 (link)].
5
Intestinal Barrier Dysfunction Regulation
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The main reagents used included PF (purity ≥ 99%, S31585) (Shanghai YuanYe Biotechnology Co., Ltd., China), rifaximin (R126176) (Shanghai Aladdin Bio-Chem Technology Co., Ltd., China), enzyme-linked immunosorbent assay (ELISA) kits of D-lactate (E02D0015), interleukin-1β (IL-1β; E02I0010), transforming growth factor-β (TGF-β; E02T0058), and tumor necrosis factor-α (TNF-α; E02T0008) (Shanghai BlueGene Biotech Co., Ltd., China), nuclear factor kappa B (NF-κB) p65 antibody (10745-1-AP) (Proteintech, Japan), phosphorylated (p-)NF-κB p65 antibody (#3033) (CST, USA), zonula occludens-1 (ZO-1) antibody (61-7300) and occludin antibody (71-1500) (Thermo Fisher Scientific Co., Ltd., China), total RNA extraction kits (#DP419), complementary DNA (cDNA) reverse transcription kits (KR116-02) and SYBR amplification kits (#P205-02) (Tiangen Biotech [Beijing] Co., Ltd., China).
The main instruments included high-performance universal benchtop refrigerated centrifuge (Thermo Fisher Sorvall ST16R; Thermo Scientific, USA), multi-function microplate reader (Thermo Scientific), optical microscope (Nikon ECLIPSE TS100; Nikon Japan), and fluorescence quantitative polymerase chain reaction (PCR) system (Bio-Rad iQ5; Bio-Rad, USA).
The main instruments included high-performance universal benchtop refrigerated centrifuge (Thermo Fisher Sorvall ST16R; Thermo Scientific, USA), multi-function microplate reader (Thermo Scientific), optical microscope (Nikon ECLIPSE TS100; Nikon Japan), and fluorescence quantitative polymerase chain reaction (PCR) system (Bio-Rad iQ5; Bio-Rad, USA).
6
Quantitative Gene Expression Analysis
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Important DEGs related to SE were selected to check their relative gene expression by qRT-PCR. RNA was extracted from the samples using a total RNA extraction kit (Tiangen, Beijing, China) according to the manufacturer’s protocol. The quality and quantity of RNA were assessed with 1.2% agarose electrophoresis and a NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA), respectively. Primers were designed using Premier 5.0 software (Created by Premier company, Toronto, Canada). The first-strand cDNA synthesis was performed using a FastQuant RT Kit (Tiangen, Beijing, China) following the manufacturer’s instructions. The relative expression of the candidate genes was calculated using a double standard curve according to the CFX96 Real-Time system (Bio-Rad, Hercules, CA, USA) by normalizing to the reference gene ACTIN according to Wang et al. (2020). All qRT-PCR reactions were performed in triplicate with at least three biological replicates. The primer information used in this experiment are shown in Table S4 .
7
Quantification of NRF2 Gene Expression
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Total RNA was extracted using a Total RNA extraction kit (Tiangen Biotech Co., Ltd.) according to the manufacturer's instructions, and perform reverse transcribed according to Prime Script RT reagent kit instructions (Takara Biotechnology Co., Ltd.). Aliquots of cDNA were subjected to qPCR analysis with SYBR Green PCR Master Mix (Takara Biotechnology Co., Ltd.) and a Real-Time PCR system equipped with a CFX 96 Connect™ Optics Module (Bio-Rad Laboratories, Inc.). Reaction program [94°C pre-denaturation for 30 sec; 40 cycles (94°C denaturation for 5 sec, 60°C annealing for 1 min)]. Primer sets specific to GAPDH (reference gene) and NRF2 were as follows: GAPDH forward, 5′-ACCACAGTCCATGCCATCAC-3′ and reverse, 5′-TCCACCACCCTGTTGCTGTA-3′; NRF2 forward, 5′-CAGTCAGCGACGGAAAGAGT-3′ and reverse, 5′-ACGTAGCCGAAGAAACCTCA-3′. Data were analyzed according to the 2−∆∆cq method as previously described (18 (link)).
8
Quantifying mRNA Expressions via qRT-PCR
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To determine mRNA expressions, total RNA was extracted from cells by the total RNA extraction kit (DP419, TIANGEN, China). Subsequently, the complementary DNA from each sample was synthesized by reverse transcription reactions using a reverse transcription kit (D7166, Beyotime, China). The qRT-PCR was conducted by a real-time PCR system (7300, Applied Biosystems, USA) with SYBR Green qPCR Mix (D7260, Beyotime, China) as per the guide. The primers are given in Table 1 . The expression levels of circ_0001998-1 and miR-145 were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6 expression by the 2−ΔΔCt method.
9
Ginsenoside Rb1 Bioactivity Assessment
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Ginsenoside Rb1 (purity > 99.5%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). SSD cream (1%) was used as a positive control.Anti-FGF-2, anti-PDGFR-β, anti-PDGF-BB and anti-β-actin antibodies were purchased from Abcam (Shanghai, China). A BCA protein quantification kit was purchased from BiyunTian (Shanghai, China). Electrochemiluminescence (ECL) reagent was purchased from Millipore (Billerica, MA, USA). A total RNA extraction kit was purchased from Tiangen (Beijing, China). A RevertAid First Strand cDNA Synthesis Kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). RT-PCR Master Mix was purchased from Vazyme (Nanjing, China).The various other chemicals used were of analytical grade and commercial origin.
10
Quantifying lncRNA Expression via qRT-PCR
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Total cellular RNA was extracted with TRIzol reagent (Invitrogen) and the Total RNA Extraction Kit (TIANGEN Biotech), the concentration of which was measured with a spectrophotometer. Quantitative real-time PCR (qRT-PCR) was performed with PrimeScript RT Master Mix (TaKaRa) according to the manufacturer’s protocol. Each cDNA sample was denatured at 95 °C for 5 min and amplified for 35 cycles of conditions including 15 s at 98 °C, 30 s at 58 °C, and 30 s at 72 °C with a LightCycler 96 (Roche). The mRNA expression level of each target gene was normalized to the respective GAPDH and analyzed using LightCycler® 96 Application Software (Roche). The qRT-PCR primers are listed in Supplementary Table 3 . Notably, five pairs of qRT-PCR primers for the newly identified lncRNAs by RNA-seq were designed and applied. The suitable primers were screened with stable results from three independent experiments and are listed in Supplementary Table 3 . Northern blotting was performed using a NorthernMax Kit (Ambion) with biotin-labeled probes, the sequences of which are shown in Supplementary Table 3 . The viral load of HTNV was quantified based on the qRT-PCR and absolute quantitative PCR.
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