The effect of the TCM on endogenous estrogen-regulated gene expression was assessed by determining the mRNA levels of GREB1, pS2, RIP140, RARα and PR in MCF-7 cells using RT-PCR. For this purpose, MCF-7 cells were treated for 24 hours with either 10 nM E2 or a 0.3% dilution of the TCM. At the end of the treatment, RNA was extracted from the cells using the RNeasy RNA isolation kit (Qiagen, Courtaboeuf, France). For RNA extractions, two independent cultures were performed per condition. Reverse transcription was done on 1 μg total RNA using random hexamers and SuperScript™ II reverse transcriptase (Invitrogen) in a reaction solution which was diluted ten times for amplification. The mRNA levels of GREB1, pS2, RIP140, RARα, and PR were quantified by RT-PCR using SYBR® Green reagents in a LightCycler® RT-PCR System (Roche SAS, Boulogne-Billancourt, France). The expression levels of each gene were normalized to that of the housekeeping 28S gene, and quantified in relative units using qBasePLUS[26 ]. Determinations were made in duplicate.
Lightcycler rt pcr system
Manufactured by Roche
Sourced in United States
The LightCycler RT-PCR System is a real-time PCR instrument designed for quantitative gene expression analysis. It provides precise and reliable detection and quantification of target nucleic acid sequences.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rneasy rna isolation kit, by Qiagen (1 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Tiangen Biotech (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Superreal premix plus, by Tiangen Biotech (1 mentions)
Fastking rt kit with gdnase, by Tiangen Biotech (1 mentions)
4 protocols using lightcycler rt pcr system
1
Estrogen-Regulated Gene Expression in MCF-7 Cells
2
Quantifying Gene Expression in Rat Liver
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RT-PCR was performed in the light cycler RT-PCR system (Roche Diagnostics, Mannheim, Germany). Each reaction contained, 2μL of cDNA (10-fold diluted), 0.5 μL of 5 mM solutions of each of the forward and reverse primers, and 7.5 μL of 2x SYBR green master mix in a total volume of 15 μL. Cyclophilin A (cyclo A) gene was used as a housekeeping gene for normalization. Primer pairs for each gene were designed using Allel ID software. The primer sequences used shown in Table 1 . All amplification reactions were performed in duplicate in each run. 2-ΔΔCT method was used to calculate the relative expression ratio (fold changes) of mRNAs in the liver tissue of test rats to those of control animals after normalizing with Cyclo A.
3
Quantitative Analysis of Lung mRNA
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Total RNA of lung tissue was extracted using TRIzol based on the manufacturer's instructions (TIANGEN, USA). 2 μg of isolated RNA was reverse-transcribed into cDNA using a cDNA synthesis kit (Thermo Fisher, USA) based on the manufacturer's protocols. Then, the qPCR was carried out by using the SYBR GREEN Master Mix (Thermo Fisher, USA) in a LightCycler RT-PCR System (Roche Diagnostics, USA). The 2–ΔΔCt method was used to quantify the mRNA expression levels. The primers are shown in Supplementary File Table S1 .
4
Total RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted using TRIzol reagent (Invitrogen). The purity and concentration of RNA were determined by measuring the ratio of absorbance at 260/280 nm. The reverse transcription and qRT-PCR were performed using the FastKing RT Kit (With gDNase) (TIANGEN, Beijing, China) and the SuperReal Premix Plus (SYBR Green) kit as per the company’s protocol, using the respective RNA as the templates. Calculation of Ct (threshold cycle) values for the amplification curves were achieved after subtracting GAPDH values for normalization. USA). The primer sequences used for qRT-PCR are listed in Table 1 . The reaction was started with denaturation at 95 °C for 15 min, and then the cDNA was amplified for 45 cycles with the denaturation at 95 °C for 10 s, annealing at 55 °C for 30 s and extension at 72 °C for 32 s. Each sample was amplified in triplicates on the LightCycler RT-PCR System (Roche 480, New York, USA).
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