The total RNA from the mammary tissues or cells was extracted using TriZol solution (TransGene, Shanghai, China) according to the manufacturer's instructions. The assessment of the quantity and quality of the total RNA was conducted by a spectrophotometer (NanoDrop Technologies, Wilmington, DE). The first‐strand cDNA was generated from 3 µg total RNA using TransScript II First‐Strand cDNA Synthesis SuperMix (TransGene). Real‐time quantitative PCR (RT‐qPCR) was carried out to examine the levels of genes by iQ5 light cycler (Bio‐Rad, Hercules, CA) in 20 µL reactions. Finally, the expression of each gene was normalized to GAPDH. The primers used are listed in Table S1 .
Trizol solution
Manufactured by Transgene
Sourced in China
TriZol solution is a reagent used for the isolation and purification of total RNA from various biological samples. It is a single-phase solution containing phenol and guanidine isothiocyanate, which effectively lyses cells and denatures nucleoproteins to release RNA. The solution maintains the integrity of the RNA during the extraction process, allowing for the recovery of high-quality, intact RNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Transscript 2 first strand cdna synthesis supermix, by Transgene (5 mentions)
Rna easy kit, by Transgene (3 mentions)
Iq5 lightcycler, by Bio-Rad (2 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (1 mentions)
Primer premier 5, by Premier Biosoft (1 mentions)
Transstart probe qpcr supermix, by Transgene (1 mentions)
Abi steponeplus pcr system, by Thermo Fisher Scientific (1 mentions)
5 protocols using trizol solution
1
Mammary Tissue RNA Extraction and Gene Expression Analysis
2
Mammary Tissue mRNA Extraction and Analysis
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Total mRNA was extracted from mammary tissue and cells by using TriZol solution (TransGene, Shanghai, China) and the RNA Easy Kit (TransGene) according to the manufacturer’s instructions. Total RNA concentration was measured by a spectrophotometer (ND 2.0; Nano Drop Technologies, Wilmington, DE, USA), and 3 µg of total mRNA was reverse-transcribed into cDNA using TransScript II First-Strand cDNA Synthesis SuperMix (TransGene). Quantitative primers were designed based on the sequences in the National Center of Biotechnology Information Database and synthesized by Sangon Biotech (Shanghai, China). The primers are listed in File S1 . Quantitative real-time was performed with an iQ5 light cycler (Bio-Rad, Hercules, CA, USA) in 20-µL reactions. GAPDH was used as a reference gene.
3
Quantitative RNA Expression Analysis
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Tissue samples were repeatedly grinded to a fine powder with liquid nitrogen using a mortar and pestle, and then the sample was transferred to 1.5 ml non-enzyme EP tube. All samples were respectively resuspended in ice-cold TriZol solution (TransGene); samples were passed through aribonuclease-free 20-gauge needle 10-20 times for disruption. Total mRNA extraction was performed with the RNA Easy Kit (TransGene) according to the manufacturer's instructions, and concentration was measured with a spectrophotometer (BioTek, Winooski, VT), and 1000 ng of RNA was reverse-transcribed into cDNA with the TransScript II First-Strand cDNA Synthesis Super Mix (TransGene).
All PCR primers were synthesized by Sangon Biotech (Shanghai, China). The primers are listed in Supplementary TableS1 . RT-qPCR was done on ABI Step One Software System (ABI, Foster City, CA). PCR conditions were as follows: 95 °C for 30 seconds, then 40 cycles of denaturation at 95 °C for 5 seconds, and finally at 60 °C for 30 seconds. Primer specificity was estimated by melting curves. Standard curves were generated with reference cDNA to determine the starting quantity of mRNA in all samples. Comparative Ct method was employed to quantify normalized specific gene expression relative to the calibrator. Data were expressed as relative gene expression = 2 -ΔΔCt, and GAPDH served as an internal control.
All PCR primers were synthesized by Sangon Biotech (Shanghai, China). The primers are listed in Supplementary Table
4
RNA Extraction and RT-qPCR Analysis
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Tissues samples were completely grinded and then transferred to 1.5 ml non-enzyme EP tube, and cells were regularly harvested into EP tube. Total RNA was extracted from tissues and cells respectively using TriZol solution (TransGene, Beijing, China) in accordance with the manufacturer's instructions. RNA was reverse-transcribed into cDNA with the TransScript II First-Strand cDNA Synthesis Super Mix (TransGene). The RT-qPCR procedure was performed on ABI Step One Software System (ABI, Foster City, CA, USA) using SYBR Premix ExTaq II kits (Takara, Tokyo, Japan). For RT-PCR, PCR product was separated by 1% agarose gel. All PCR primers were designed by Primer Premier 5 (PREMIER Biosoft international, Palo Alto, CA, USA) and synthesized by TsingKe Biological Technology (Xian, Shaanxi, China). The primers are listed in Table S1 . GAPDH or U6 were used as internal control.
5
Quantitative Gene Expression Analysis
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Cells were washed and re-suspended in ice-cold TriZol solution (TransGene, Shanghai, China). Total mRNA extraction was performed using an RNA Easy Kit (TransGene), according to the manufacturer's instructions. The concentration of mRNA was measured using a spectrophotometer (ND 2.0; NanoDrop Technologies, Wilmington, DE, USA). One microgram of total RNA from each sample was reverse-transcribed into cDNA using a TransScript II First-Strand cDNA Synthesis SuperMix (TransGene).
Real-time PCR was performed with an ABI StepOnePlus PCR system (Applied Biosystems, Foster City, CA, USA), using the TransStart Probe qPCR SuperMix (TransGene) and the reaction system recommended by the manufacturer. Specific forward and reverse PCR primers were purchased from Sangong Biotech (Sangong Biotech, Shanghai, China), and are listed in Table 1.GAPDH was used as an internal control in all reactions. The PCR conditions were as follows: activation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s, extension at 60 °C for 30 s. Melt curves were generated to evaluate the specificities of the primers. The comparative cycle threshold (Ct) method was used to quantify normalized target gene expression relative to the calibrator. Data are shown as the relative gene quantity (RQ) =2 -ΔΔCt .
Real-time PCR was performed with an ABI StepOnePlus PCR system (Applied Biosystems, Foster City, CA, USA), using the TransStart Probe qPCR SuperMix (TransGene) and the reaction system recommended by the manufacturer. Specific forward and reverse PCR primers were purchased from Sangong Biotech (Sangong Biotech, Shanghai, China), and are listed in Table 1.GAPDH was used as an internal control in all reactions. The PCR conditions were as follows: activation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s, extension at 60 °C for 30 s. Melt curves were generated to evaluate the specificities of the primers. The comparative cycle threshold (Ct) method was used to quantify normalized target gene expression relative to the calibrator. Data are shown as the relative gene quantity (RQ) =2 -ΔΔCt .
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