The total RNA was extracted from BMSCs culture on day 7 using RNAEX reagent (Accurate Biotechnology, Hunan, China). Extracted 500 ng total RNA from each sample was reverse transcribed to cDNA using Evo M-MLV RT Premix for qRT-PCR (Accurate Biotechnology, Hunan, China). The reverse-transcribed cDNA was subjected to RT-qPCR (SYBR Green Premix Pro Taq HS qPCR Kit, Accurate Biotechnology, Hunan, China) using the following cycling conditions: 95°C for 10 min (initial denaturation), 40 cycles of 95°C for 15 s and 60°C for 60 s and then 60°C for 5 min for the terminal extension. The temperature was increased by 1 degree every 20 s to obtain the melting curve. The relative quantitative ΔΔCt method was used to determine the fold change of expression. The expressions of osteogenic gene RUNX2, ALP, OCN and BMP2 were analyzed by RT-qPCR. GAPDH was used as an internal control to normalize the expression of specific genes. The primer sequences used for RT-qPCR are listed in Table 1 .
Rnaex reagent
Manufactured by Accurate Biology
Sourced in China
RNAEX reagent is a ready-to-use solution designed for the isolation and purification of total RNA from various biological samples. It contains a proprietary blend of chaotropic agents, detergents, and buffers that facilitate the lysis of cells and the inactivation of RNases, allowing for the efficient extraction and recovery of high-quality RNA.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green premix pro taq hs qpcr kit, by Accurate Biology (7 mentions)
Rnase r, by Illumina (2 mentions)
Evo mmlv rt premix for qrt pcr, by Accurate Biology (1 mentions)
Rt pcr system, by Takara Bio (1 mentions)
Rt pcr system, by Bio-Rad (1 mentions)
Quantitative real time polymerase chain reaction, by Takara Bio (1 mentions)
Evo m mlv rt premix, by Accurate Biology (1 mentions)
Lc ms grade formic acid, by Merck Group (1 mentions)
Lc ms grade acetonitrile, by Thermo Fisher Scientific (1 mentions)
Milli q system, by Merck Group (1 mentions)
Evo m mlv rt mix kit, by Accurate Biology (1 mentions)
Occludin, by Santa Cruz Biotechnology (1 mentions)
Evo m mlv mix kit, by Accurate Biology (1 mentions)
Quantstudio 5 system, by Thermo Fisher Scientific (1 mentions)
Hieff qpcr sybr green master mix, by Yeasen (1 mentions)
Steadypure universal rna extraction kit, by Accurate Biology (1 mentions)
Quantstudio 6 flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480ii pcr instrument, by Roche (1 mentions)
Evo m mlv rt premix for qpcr, by Accurate Biology (1 mentions)
8 protocols using rnaex reagent
1
Osteogenic Gene Expression in BMSCs
2
Quantitative Analysis of RNA Expression
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Total RNA was extracted from primary chondrocytes or cartilage tissues using RNAEX reagent (Accurate Biotechnology, Hunan, China), according to the manufacturer’s instructions. Specific mRNAs were qualified using SYBR® Green Premix Pro Taq HS qPCR kit (Accurate Biotechnology, Hunan, China), according to the manufacturer’s instructions. The levels of lncRNAs and mRNAs were normalized to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primers used are shown in Supplementary Table S3 .
3
RNA Extraction and RT-qPCR Quantification
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The total RNA was extracted from tissues using RNAEX reagent (Accurate Biotechnology, China). It was reverse transcribed into cDNA by using Evo M-MLV RT premix (Accurate Biotechnology, China) and quantitative real-time polymerase chain reaction (RT-PCR) system (TaKaRa, Japan). Then a SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology, China) was used for PCR. The PCR liquid volume was 20 μL. Quantitative PCR was performed using an RT-PCR system (BioRad, Singapore). At 95℃, 40 cycles were amplified after 90 s of initial denaturation, 10 s desaturation at 95℃, and 34 s of extension at 60℃. GAPDH was used as the reference gene for calculation. And the primer sequences are shown in Table 1 .
4
Sijunzi Decoction Protocol for Colitis
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The Chinese medicinal materials used in Sijunzi decoction were sourced from the Bozhou Medicinal Materials Market in Anhui Province, China, and met the standards set by the Pharmacopoeia of the People’s Republic of China. Interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were purchased from Meimian Biotechnology (Yancheng, Jiangsu Province, China). Dextran sulfate sodium (DSS, molecular weight: 36–50 kDa) was obtained from Santa Ana MP Biomedicals in California, USA. Occludin and ZO-1 monoclonal antibodies were bought from Santa Cruz Biotechnology companies in California, USA. The water and methanol used in the experiment were of HPLC grade. Ultrapure water was obtained by filtration of distilled water using a Milli-Q system (Millipore, USA). LC–MS-grade acetonitrile was purchased from Thermo Fisher Scientific (Fair Lawn, New Jersey, USA), and LC–MS-grade formic acid was purchased from Sigma-Aldrich (St. Louis, Missouri, USA). RNAex Reagent, Evo M-MLV RT Mix Kit, and SYBR@ Green Premix Pro Taq HS qPCR Kit were acquired from Accurate Biotechnology (Changsha, China).
5
Quantification of circRNAs, mRNAs, and miRNAs in Chondrocytes
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Total RNA was extracted from primary chondrocytes using RNAEX reagent (Accurate Biotechnology, Hunan, China) according to the manufacturer's instructions. Reverse transcription of mRNAs or miRNAs to cDNA was performed using total RNA, with kits from Accurate Biotechnology (Hunan, China) or Sangon (Shanghai, China), respectively. Specific circRNAs or mRNAs were quantified with SYBR® Green Premix Pro Taq HS qPCR kit (Accurate Biotechnology, Hunan, China), and normalized to ACTB. Specific miRNAs were quantified with the MicroRNAs qPCR kit (Sango, Shanghai, China), and normalized to U6. The 2-ΔΔCt method was used to calculate the relative expression. All experiments were independently performed in triplicate. All primers are listed in Table S2
6
Quantification of Thyroid Cancer miRNAs
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Total RNAs of thyroid cancer tissues and cells were extracted by using RNAEX reagent (Accurate Biotechnology, Hunan) with instructions of the manufacturer. RNase R (Epicenter Technologies) was used for 15 min at 37°C when RNase R treatment was necessary. The first strand of cDNA was synthesized with Evo M-MLV Mix Kit (Accurate Biotechnology, Hunan). Particularly, the first strand of cDNA of miR-361–3p and miR-451a was synthesized using the stem-loop method, while the first strand of cDNA of U6 was synthesized with a gene-specific primer. SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology, Hunan) was used for subsequent qRT-PCR assay on QuantStudio 5 system (ThermoFisher Scientific, USA). The relative expression levels were analyzed using the 2−ΔΔCt method and normalized by beta-actin or U6. The sequences of primers involved in this study were shown in Table 1 .
7
Total RNA Extraction and qPCR Analysis
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RNAEX reagent (Accurate Biotechnology, Hunan, China) and the SteadyPure Universal RNA Extraction Kit (Accurate Biotechnology, Hunan, China) were used to extract total RNA from cells according to the manufacturer’s instructions. For mRNA analysis, Evo M-MLV RT Premix for quantitative PCR (qPCR) (Accurate Biotechnology, Hunan, China) and Hieff qPCR SYBR Green Master Mix (catalog no. 11201, Yeasen, Shanghai, China) were used according to the manufacturer’s instructions, and the reactions were then measured using a QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific) according to the manufacturer’s instructions. The reactions were normalized to the mRNA housekeeping gene β-actin. Primers were obtained from Tsingke (Beijing, China).
8
Quantification of Circular RNAs and mRNAs
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Total RNA from cells and tissues was extracted using the RNAEX reagent (Accurate Biotechnology, Hunan) according to the manufacturer’s instructions. For RNase R treatment, 2 mg of total RNA was incubated with or without 3 U/mg RNase R (Epicenter Technologies) for 15 min at 37°C. For circRNA and mRNA analyses, Evo M-MLV RT Premix for qPCR (Accurate Biotechnology, Hunan) and SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology, Hunan) were used, and the reactions were subsequently measured using a Roche LightCycler 480II PCR instrument (Basel, Switzerland) in accordance with the manufacturer’s recommended protocols. The reactions were normalized to the miRNA housekeeping gene U6 or the mRNA housekeeping gene β-actin. For absolute quantification, standards were generated and diluted. Standard curves between CT values and log10(copy numbers) were constructed. The primers were obtained from RiboBio (Guangzhou, China).
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