Total RNA from mouse brain tissues was extracted using RNAzol (Thermo Fisher). RNA were then reverse transcribed into cDNA using the TIANScript II RT Kit (Tiangen). The reaction mix was combined with cDNA, SYBER green mix, and corresponding primers. PCR reactions were performed in duplicate for each sample and the average Ct values were used to calculate the mRNA expression levels (CFX96 Touch Real-Time PCR Detection System, Bio-Rad). Ct values of miR-339-5p were normalized to that of U6.
Rnazol
Manufactured by Thermo Fisher Scientific
Sourced in United States
RNAzol is a reagent designed for the isolation and purification of total RNA from a variety of biological samples. It is a single-step method that ensures the integrity of the extracted RNA.
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Lab products found in correlation
Sybr green pcr master mix, by Thermo Fisher Scientific (18 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (6 mentions)
Steponeplus, by Thermo Fisher Scientific (4 mentions)
Dnase 1, by Thermo Fisher Scientific (3 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Rneasy kit, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Superscript 2, by Thermo Fisher Scientific (2 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (2 mentions)
Sybr green, by Roche (2 mentions)
Tianscript 2 rt kit, by Tiangen Biotech (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Quantscript rt kit, by Tiangen Biotech (1 mentions)
Dnase free rnase, by Merck Group (1 mentions)
Sensifast cdna synthesis kit, by Meridian Bioscience (1 mentions)
Steponeplus cycler, by Thermo Fisher Scientific (1 mentions)
Sensifast sybr hi rox kit, by Meridian Bioscience (1 mentions)
Direct zol rna miniprep plus kit, by Zymo Research (1 mentions)
Oligo dt 18, by Thermo Fisher Scientific (1 mentions)
Forward and reverse primer, by Thermo Fisher Scientific (1 mentions)
27 protocols using rnazol
1
Quantifying miR-339-5p Expression in Mouse Brain
2
Quantitative Gene Expression Analysis
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Skin, liver, and spleen tissues were homogenized and total RNA was extracted with RNAzol (Thermo Fisher Scientific). Extracted total RNA was diluted to 1 μg/μL, and 5 μL was used for reverse transcription to synthesize cDNA. Two microliters of cDNA was mixed with 10 μL SYBR Green PCR Master Mix and 1 μL of each primer (Thermo Fisher Scientific, Table 2 ) for PCR reaction under the following conditions: 95°C for 60 s, followed by 40 cycles of 95°C for 15 s, 55°C for 30 s, and 72°C for 35 s, and a final 95°C for 30 s, followed by 55°C for 35 s. GAPDH was used as internal reference, and relative gene transcript levels were calculated using 2−ΔΔCt method [18 (link)].
3
Quantitative PCR Analysis of Liver Gene
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The liver tissue of mice was crushed and the total RNA was extracted by RNAzol (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and diluted to 1 µg/µL. The diluted total RNA solution of 5 µL was reverse transcribed with a kit (Quantscript RT kit, Tiangen Biotech Co., Ltd., Beijing, China), and the DNA template was obtained. For the qPCR, 2 µL of DNA template was mixed with 10 µL of SYBR Green PCR Master Mix (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1 µL of (100 nmol/mL) forward and reverse primers (Table 1 ), reacted at 95 °C for 60 s, then 40 cycles were performed at 95 °C for 15 s, 55 °C for 30 s, and 72 °C for 35 s. Finally, the gene expression was detected at 95 °C for 30 s, and 55 °C for 35 s by a real time PCR instrument (SteponePlus, Thermo Fisher Scientific, Inc., Waltham, MA, USA). GAPDH was used as an internal reference, and the relative gene expression was calculated using the 2−ΔΔCt method [18 (link)].
4
Mouse Small Intestine RNA Extraction and Analysis
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The RNA of the small intestine tissues of mice was extracted using the extractor RNAzol (Thermo Fisher Scientific). Total RNA was digested using RNase‐free water at 37°C for 15 min. The RNA was then purified using the RNeasy kit (Thermo Fisher Scientific) at the concentration of 1 μg/μl. The RNA template was synthesized to cDNA at 37°C for 120 min, 99°C for 4 min, and 4°C for 3 min. The mRNA expression levels (Table 4 ) were measured by real‐time quantitative PCR (qPCR; StepOnePlus, Thermo Fisher Scientific) to conduct 40 cycles at 95°C for 3 min (predenaturation), 95°C for 10 s (denaturation), 57°C for 30 s (annealing), and 72°C for 15 s (extension). The relative mRNA expression levels were ultimately calculated using the 2−ΔΔCr formula (Qian, Song, Sun, et al., 2018 ; Qian, Song, Yi, et al., 2018 ).
5
Quantitative RT-PCR Analysis of Mouse Liver and Spleen
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Total RNA of liver and spleen tissues from the mice was extracted using RNAzol (Thermo Fisher Scientific, Inc.). The concentration of the extracted total RNA was diluted to 1 μg/μl, and 5 μl of the diluted total RNA solution was used in the assay. To obtain a cDNA template, extraction and reverse transcription were performed according to the manufacturer's instructions. The following procedure was performed for 40 cycles: 2 μl of cDNA template and 10 μl of SYBR Green PCR Master Mix, 1 μl of upstream and downstream primers (Thermo Fisher Scientific) were mixed and reacted for 60 s at 95°C, and 15 s at 95°C, 30 s at 55°C, and 33 s at 72°C, respectively. Finally, they were both tested for 30 s at 95°C and 35 s at 55°C, internally referred to GAPDH. The 2−ΔΔCt method was used to calculate expression (Liu, Zhang, et al., 2019 ).
6
Myocardial Cell RNA Isolation and qPCR Analysis
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Total RNA was extracted from myocardial cells using RNAzol (Thermo Fisher Scientific, Inc.), and DNase RNase-free (Sigma-Aldrich; Merck KGaA) was used to digest total RNA at 37°C for 15 min. An RNeasy kit (Thermo Fisher Scientific, Inc.) was then used to purify RNA and the concentration was adjusted 1 µg/µl. A total of 2 µg RNA was used as the template to synthesize cDNA at 37°C for 120 min, 99°C for 4 min and 4°C for 3 min. Followed by, qPCR was performed to amplify the expression of IL-6, TNF-α, IL-10, IL-17A, B-cell lymphoma (Bcl)-2, P53, p38 MAPK and NF-κB, with β-actin as a control (Table I ). Thermocycling conditions were as follows: 95°C for 2 min, followed by 35 cycles of 95°C for 20 sec, 55.8°C for 30 sec and 72°C for 30 sec, followed by a final extension at 72°C for 5 min. Following RT-qPCR, agarose electrophoresis with 1% ethidium bromide was used to check the amplified PCR products. Gene expression was quantified using TaqMan Gene Expression Assays (Thermo Fisher Scientific, Inc.). Relative mRNA expression changes were calculated using the 2−ΔΔCq method (19 (link)).
7
Quantitative Real-Time PCR Gene Expression Analysis
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Stomach specimens were homogenized. Total RNA was extracted using RNAzol (Thermo Fisher Scientific, Waltham, MA, USA), and the concentration was diluted to 1 μg/μL. Thereafter, 5 μL RNA was reverse transcribed to prepare DNA. Approximately 2 μL of DNA was combined with 1 μL of the forward primer, 1 μL of the reverse primer (Table 1 ), and 10 μL of SYBR Green PCR Master Mix and amplified at 95 °C for 60 s, followed by 40 cycles at 95 °C for 15 s, 55 °C for 30 s, and 72 °C for 35 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control. The relative expression of each target gene was calculated by the 2−ΔΔCt method [13 (link)].
8
qPCR Analysis of Receptor Tyrosine Kinases
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For analysis of EPHA7, EPHB3, PLXDC1 and PLXDC2 expression by qPCR, 1x106 cells were harvested in RNAzol (Thermo Fisher) and RNA was isolated using the Direct-zol RNA Miniprep Plus Kit (Zymo) according to the manufacturer’s instructions. 1μg total RNA was used for cDNA synthesis using the SensiFAST cDNA Synthesis Kit (Bioline) according to manufacturer’s instructions. qPCR was performed on a StepOne Plus cycler (Thermo Fisher Scientific) in 20μl reactions using the SensiFAST SYBR Hi-ROX Kit (Bioline) (cycling conditions: 2min initial denaturation at 95°C, 40 cycles: 95°C for 5s and 65°C for 25s). Relative expression was calculated based on ΔCt to GAPDH. Samples were analyzed in technical triplicates, only samples with amplification in all triplicates were scored as positive. See S2 Table for a complete list of primers.
9
Extraction and Analysis of Intestinal RNAs
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Small intestine tissue from mice were crushed, and general RNAs of colon tissue were extracted by RNAzol (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The total RNA concentration was diluted to 1 lg/lL. Then, 2 lL of diluted general RNA extraction solution was added to 1 lL each of oligodT18, RNase, dNTP, and MLV enzymes (Thermo Fisher Scientific, Inc.) and 10 lL of 5 • buffer to obtain cDNA at 37°C for 120 min, 99°C for 4 min, and 4°C for 3 min. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to amplify c-Kit, stem cell factor (SCF), transient receptor potential cation channel subfamily V member 1 (TRPV1), glial cell-derived neurotrophic factor (GDNF), and NO synthase (NOS) (Thermo Fisher Scientific, Inc.) mRNA expression (Table 1), and the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as the reference. Finally, electrophoresis on 1% agar gel was performed to measure amplified PCR products. 8 ImageJ 1.44 software was used to conduct semi-quantitative analysis of the results.
10
Quantitative Gene Expression Analysis in Mouse Tissues
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The liver and spleen tissues of the mice were homogenized, and then RNAzol (Thermo Fisher Scientific Inc.) was used to extract total RNA from the tissues; the extracted RNA was diluted to a total concentration of 1 μg/μL. Then, 5 μL of the diluted total RNA solution was applied for reverse transcription according to the reverse transcription kit method to obtain a cDNA template. After 2 μL of cDNA template was mixed with 10 μL of SYBR Green PCR Master Mix and 1 μL of upstream and downstream primers (Thermo Fisher Scientific Inc.; Table 1), the reaction proceeded at 95°C for 60 s, followed by 40 cycles of 95°C for 15 s, 55°C for 30 s, and 72°C for 35 s. Finally, detection was carried out at 95°C for 30 s and at 55°C for 35 s using glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as the internal reference (StepOnePlus, Thermo Fisher Scientific Inc.). The 2 -ΔΔCt method was used to calculate relative gene expression (Manini, 2010) .
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