Based on results of MTT assay, after treating the test cells with the combination of 20 nM EE and 90 nM LNG, and control cells with the normal medium for 48h, the total RNA was isolated using RNeasy Mini, RNA isolation kit (Qiagen, Germany) according to manufacturer’s instructions. RNA concentration was measured by Nanodrop 2000c spectrophotometer (Thermo Scientific, USA), and cDNA were synthesized by cDNA Synthesis Kit (Bio FACT, Daejeon, South Korea). The changes in mRNA expression of MKI67, BAX, Bcl2 genes and Beta-2-microglobulin (β2M), as internal control, were estimated by quantitative reverse transcriptase PCR (qRT-PCR) in a rotor gene 6000 Corbett (Corbett Research, Sydney, Australia) detection system SYBR GREEN® (nonspecific DNA-binding factors). The primer sequences are provided in
Relative expression software tool
Manufactured by Qiagen
Sourced in Germany
The Relative Expression Software Tool is a bioinformatics application designed to analyze and visualize gene expression data. The software allows users to calculate the relative expression levels of genes across different samples or experimental conditions.
Automatically generated - may contain errors
Lab products found in correlation
Corbett rotor gene 6000, by Qiagen (1 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rneasy mini, by Qiagen (1 mentions)
Cdna synthesis kit, by Biofact (1 mentions)
Primer express 2, by Thermo Fisher Scientific (1 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Rest 2009, by Qiagen (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Iq5 cycler, by Bio-Rad (1 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Sybr green qpcr master mix, by MedChemExpress (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Relative expression software tool 2009, by Qiagen (1 mentions)
6 protocols using relative expression software tool
1
Quantifying Expression of Cell Proliferation and Apoptosis Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
Based on results of MTT assay, after treating the test cells with the combination of 20 nM EE and 90 nM LNG, and control cells with the normal medium for 48h, the total RNA was isolated using RNeasy Mini, RNA isolation kit (Qiagen, Germany) according to manufacturer’s instructions. RNA concentration was measured by Nanodrop 2000c spectrophotometer (Thermo Scientific, USA), and cDNA were synthesized by cDNA Synthesis Kit (Bio FACT, Daejeon, South Korea). The changes in mRNA expression of MKI67, BAX, Bcl2 genes and Beta-2-microglobulin (β2M), as internal control, were estimated by quantitative reverse transcriptase PCR (qRT-PCR) in a rotor gene 6000 Corbett (Corbett Research, Sydney, Australia) detection system SYBR GREEN® (nonspecific DNA-binding factors). The primer sequences are provided inTable-1 . Alterations of gene expression of KI67, BAX, and Bcl2 compared with β2M were normalized by LinReg software (Linreg version 2012.1, Amsterdam, the Netherlands). Expression levels of the mentioned gens were quantified by REST program (Relative Expression Software Tool, Qiagen, Germany).
+ Expand
Based on results of MTT assay, after treating the test cells with the combination of 20 nM EE and 90 nM LNG, and control cells with the normal medium for 48h, the total RNA was isolated using RNeasy Mini, RNA isolation kit (Qiagen, Germany) according to manufacturer’s instructions. RNA concentration was measured by Nanodrop 2000c spectrophotometer (Thermo Scientific, USA), and cDNA were synthesized by cDNA Synthesis Kit (Bio FACT, Daejeon, South Korea). The changes in mRNA expression of MKI67, BAX, Bcl2 genes and Beta-2-microglobulin (β2M), as internal control, were estimated by quantitative reverse transcriptase PCR (qRT-PCR) in a rotor gene 6000 Corbett (Corbett Research, Sydney, Australia) detection system SYBR GREEN® (nonspecific DNA-binding factors). The primer sequences are provided in
2
Optimized qRT-PCR Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For qRT-PCR analysis, cDNA was synthesized using SuperScript (Invitrogen, Carlsbad, CA, USA) with oligo(dT)s, using 500 ng of total RNA for each sample. Three biological replicates and three technical replicates were used for each time point validation (nine replicates in total). Primers were designed based on SAS sequences using the Primer Express 2.0 (Applied Biosystems, Foster City, CA, USA) software, and only primers with an amplification efficiency between 90 and 110% were used (Supplementary Table S14 ). Reaction mixtures consisted of 5 μL of 1.5 μM primer mix, 1 μL of cDNA (diluted 1:10) and 6 μL of Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA). Reactions were performed on 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). For primer SCSGLR1045D05.g, primer concentration was reduced to 312.5 nM. Expression ratio was estimated using REST 2009 (Relative Expression Software Tool, Qiagen, Hilden, Germany) as described in [107 (link)]. The endogenous genes for each tissue were identified using the geNorm software [108 (link)] (Supplementary Table S15 ).
3
Quantifying Cotton Fiber Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted from cotton fiber using the method reported by Iqbal et al.18 and reverse transcribed chemically into cDNA using cDNA Synthesis Kit (Fermentas, cat#1622). Expression analysis of the GhACTIN1 gene at the mRNA level was done through quantitative real-time PCR in transgenic and non-transgenic control cotton fiber. The relative quantification of gene expression was done using (BIO-RAD) iQ5 Cycler. Data normalization was done using GAPDH as internal control and non-transgenic/wild-type cotton plants as negative control. All samples were analyzed in triplicate with the primers: Act-F 5’-GGCAGATGGTGAGGCTATTC-3’ & Act-R 5’-CTTGCTTTGGGCTTCATCTC-3’. After completing Rt-qPCR, the analyses for relative gene expression were performed by Qiagen tool, Relative Expression Software Tool REST abbreviated as REST (http://rest.gene-quantification.info/ ) and ANOVA (Analysis of variance) was performed to compare the transgenic and non-transgenic control cotton plants for expression of the transgene.
4
Quantitative Real-Time PCR for TRAF3IP2 and TGF-β1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was prepared from cultured cells using TriZol reagent (Invitrogen) according to the manufacturer’s instruction [24 (link)]. Total RNA was reverse transcribed with random primers and oligo dT (18 mers) (Agilent Technologies). The quantitative real-time PCR reactions were performed by SYBR Green qPCR Master Mix (MedChemExpress, China) on a Rotor-GeneTM 3000 real-time PCR machine (Microspeed Biotechnology, Shanghai, China). The specific primers forTRAF3IP2 mRNA and TGF-β1 mRNA were as follows: TRAF3IP2, 5′-TGGCACCCAACAGCTTGTC-3′ (forward) and 5′-GATACAGGCCGCTGGTGATTT-3′ (reverse); TGF-β1, 5′-GGCGATACCTCAGCA ACCG-3 (forward) and 5′-CTA AGG CGA AAG CCC TCA AT-3′ (reverse). The initial denaturation was performed for 5 min at 95°C following 50 cycles of denaturation for 10 s at 95°C. Eventually, the extension was performed at 73°C for 20 s under primer-specific conditions. Comparative quantitation was performed in selected groups using the Relative Expression Software Tool (Relative Expression Software Tool, Qiagen), which is based on the Pair Wise Fixed Reallocation Randomization Test©.
5
Statistical Analysis of Experimental Groups
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The groups were compared by one-way analysis of variance (ANOVA) followed by a specific post hoc test. Statistical analysis was performed using Relative Expression Software Tool 2009 (Relative Expression Software Tool, Qiagen, Hilden, Germany), which is based on the Pair Wise Fixed Reallocation Randomization Test© (Pfaffl et al., 2002). Differences were considered statistically significant for *p < 0.05, **p < 0.01, and ***p < 0.001.
6
Dietary Effects on Lipid Metabolism
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All the data were presented as means with their standard errors.
Percentage data for survival rate, total lipid content and fatty acid composition were all arcsin-transformed before statistical analysis. Data were tested for normality and homogeneity of the variances with Levene's test. Normal distribution data were analysed by one-way ANOVA followed, when appropriate, by a Tukey's comparison of means test. Two-way ANOVA was used to determine the interaction between dietary treatments and sampling point (weight). Significant differences were accepted at P ≤ 0•05. All statistical analyses were performed using Minitab (version 16.1; Minitab Inc.). Gene expression results were analysed using the relative expression software tool (Qiagen, http://rest.gene-quantification.info/) with efficiency correction (42) to determine the statistical significance of expression ratios (gene expression fold changes) between the three treatments.
Percentage data for survival rate, total lipid content and fatty acid composition were all arcsin-transformed before statistical analysis. Data were tested for normality and homogeneity of the variances with Levene's test. Normal distribution data were analysed by one-way ANOVA followed, when appropriate, by a Tukey's comparison of means test. Two-way ANOVA was used to determine the interaction between dietary treatments and sampling point (weight). Significant differences were accepted at P ≤ 0•05. All statistical analyses were performed using Minitab (version 16.1; Minitab Inc.). Gene expression results were analysed using the relative expression software tool (Qiagen, http://rest.gene-quantification.info/) with efficiency correction (42) to determine the statistical significance of expression ratios (gene expression fold changes) between the three treatments.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!