Total RNA was extracted using Trizol reagent (Invitrogen) and reverse transcription was performed using HiScript®Q RT SuperMix (Vazyme, Nanjing, China) according to the manufacturer's instructions. qPCR was performed using AceQ® qPCR SYBR® Green Master Mix (High ROX Premixed) (Vazyme) in an ABI StepOne™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). mRNA expression levels were quantified using the 2ΔΔCT method (Applied Biosystems). The sequences of the primers used are listed in Table 1 .
2δδct method
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 2ΔΔCT method is a quantitative real-time PCR data analysis technique used to determine the relative change in gene expression levels between samples. It provides a mathematical model for analyzing the relative changes in gene expression in a simple, accurate, and efficient manner. The core function of the 2ΔΔCT method is to calculate the relative expression of a target gene in a test sample compared to a reference sample, without requiring the use of standard curves.
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Lab products found in correlation
Real time pcr system software v2, by Thermo Fisher Scientific (2 mentions)
Rneasy plus micro kit, by Qiagen (2 mentions)
Aceq qpcr sybr green master mix high rox premixed, by Vazyme (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Abi stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
Hiscript q rt supermix, by Vazyme (1 mentions)
High capacity cdna reverse transcription, by Thermo Fisher Scientific (1 mentions)
Sensi fast syber lo rox kit, by Meridian Bioscience (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Dataassist software, by Thermo Fisher Scientific (1 mentions)
7500 fast real time pcr, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase 1, by Takara Bio (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Superscript first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Sybr green 2, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Rnalater, by Qiagen (1 mentions)
7 protocols using 2δδct method
1
RNA Extraction and qPCR Analysis
2
Quantifying Host Transcriptional Response to Microbiome Modulation
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Total RNA extraction from rotavirus-infected cell lysates and/or treated with C. sorokiniana and/or B. longum was performed by the Trizol method (Life Technologies, Rockville, MD, USA). Total RNA was used as a template for cDNA synthesis (High-Capacity cDNA Reverse Transcription; Applied Biosystems, Foster City, CA, USA). Relative expression of IFN-γ, IL-10, SOCS3, STAT1, and STAT2 genes was determined by qPCR using PGK-1 as an endogenous gene (Table 1 ). Reactions were developed with the Sensi FAST SYBER Lo-ROX Kit (Bioline, London, UK), following the manufacturer’s instructions. qPCR conditions were 95 °C for 5 min, 45 cycles at 58 °C for 5 s, and 60 °C for 10 s. Gene relative expression was calculated using the 2(−ΔΔCt) method (Applied Biosystems).
3
RNA Expression Analysis in Galactosaemia
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For the RNA assays: Fold change of gene expression was quantified from raw CT scores with DataAssist software, version 3.01 using the 2ΔΔCT method (Applied Biosystems). Genes with undetermined CT scores were excluded. Two control and three galactosaemia samples with undetermined CTs for the selected normaliser genes were excluded entirely. This was likely due to insufficient PBMC pellet or excessive RNA contamination in these samples. Next, the fold change values were tested for normality of distribution with Shapiro Wilk’s test and checked for homogeneity of variance with Levene’s test.
A t-test was not suitable as most of the genes were not normally distributed. We thus opted for a Mann Whitney U test which evaluates differences between the groups irrespective of normality or variance. Type 1 errors were controlled with application of the Benjamini–Hochberg False Discovery Rate using R-software, version 3.4.0.
Spearman’s rank correlation coefficient (rs) was used to evaluate correlating data. Preparation of boxplots and scatterplots, testing of statistical differences between groups and correlation tests were conducted with SPSS software, version 24 (IBM, New York, USA).
A t-test was not suitable as most of the genes were not normally distributed. We thus opted for a Mann Whitney U test which evaluates differences between the groups irrespective of normality or variance. Type 1 errors were controlled with application of the Benjamini–Hochberg False Discovery Rate using R-software, version 3.4.0.
Spearman’s rank correlation coefficient (rs) was used to evaluate correlating data. Preparation of boxplots and scatterplots, testing of statistical differences between groups and correlation tests were conducted with SPSS software, version 24 (IBM, New York, USA).
4
qRT-PCR Analysis of TREM-1 Expression
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Total RNA of cultured aCP and pCP cells was isolated with Trizol Reagent (TaKaRa, Tokyo, Japan) according to manufacturer's protocol. After digestion with RNase-free DNase I (TaKaRa), the total amount of RNA was determined by measuring probes on a NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA), followed by reverse transcription using SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) with oligo (dT) primers. qRT-PCR with Sybr Green II (Applied Biosystems, Santa Clara, CA, USA) was employed to quantitatively assess the expression of TREM-1 in 6 aCP and 6 pCP cultured cells. All analyses were carried out with the Applied Biosystems 7500 Fast Real-Time-PCR. β-actin was used as an endogenous control for cDNA amount.
Primers for human TREM-1 were as follows: forward, 5′-GGCCACACCAACCTTCTG-3 and reverse, 5′-AGTGCCTGCCTCAATGTCTCCA-3′. Primers for actin were as follows: forward, 5′-AGAAAATCTGGCACCACACC-3′ and reverse, 5′-AGAGGCGTACAGGGATAGCA-3′. Analysis was conducted using the 2-ΔΔCT method according to the manufacturer's instructions (Applied Biosystems). All analyses were carried out in quadruplicate and evaluated statistically.
Primers for human TREM-1 were as follows: forward, 5′-GGCCACACCAACCTTCTG-3 and reverse, 5′-AGTGCCTGCCTCAATGTCTCCA-3′. Primers for actin were as follows: forward, 5′-AGAAAATCTGGCACCACACC-3′ and reverse, 5′-AGAGGCGTACAGGGATAGCA-3′. Analysis was conducted using the 2-ΔΔCT method according to the manufacturer's instructions (Applied Biosystems). All analyses were carried out in quadruplicate and evaluated statistically.
5
Real-Time qPCR of Chicken Brain
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Total RNA for each sample was extracted pooled 10 BPs using the RNeasy Plus Micro kit (catalog # 74004, QIAGEN, Venlo, Netherlands) according to the manufacturer’s protocol. DNase I treatment was performed using spin columns. RNA was reverse-transcribed using the SuperScript III First-Strand Synthesis System. Quantitative real-time polymerase chain reaction (qPCR) was performed using a StepOnePlus Real-Time PCR system. cDNA was amplified using the Power SYBR Green PCR Master Mix or TaqMan™ Fast Advanced Master Mix. The experiment was performed in triplicate. Target gene expression was normalized to hypoxanthine phosphoribosyltransferase 1 (Hprt1).112 (link) cDNA from the brain tissue of post-hatched 1-day-old chickens was used to generate standard curves for each gene. Relative quantification was performed using Real-Time PCR System Software v2.0, with the 2−ΔΔCT method (Thermo Fisher Scientific). The primers used are shown in the key resources table .
6
Quantifying Gene Expression in Chicken Brain
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Total RNA for each sample was extracted pooled 10 BPs using the RNeasy Plus Micro kit (catalog # 74004, QIAGEN, Venlo, Netherlands) according to the manufacturer’s protocol. DNase I treatment was performed using spin columns. RNA was reverse-transcribed using the SuperScript III First-Strand Synthesis System (catalog #N8080234, Thermo Fisher Scientific, Waltham, MA, USA). Quantitative real-time polymerase chain reaction (qPCR) was performed using a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was amplified using the Power SYBR Green PCR Master Mix (catalog #4367659, Thermo Fisher Scientific, Waltham, MA, USA). Target gene expression was normalized to hypoxanthine phosphoribosyltransferase 1 (Hprt1, Hassanpour et al., 2019 (link)). cDNA from the brain tissue of post-hatched 1-day-old chickens was used to generate standard curves for each gene. Relative quantification was performed using Real-Time PCR System Software v2.0, with the 2−ΔΔCT method (Thermo Fisher Scientific, Waltham, MA, USA). The following primers were used:
7
Auditory Bulla Gene Expression Analysis
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Auditory bullae were obtained from mice aged P0-P4. One bulla from each mouse was removed to RNA-later (Qiagen) and stored at -80°C until analysis. The opposite ear from each of these animals was fixed in paraformaldehyde and processed to determine the inner ear phenotype. Nine heterozygote and 9 homozygote animals were included in gene expression analysis. The qPCR analysis was performed blind to the phenotype. RNA was extracted using an RNeasy kit (Qiagen), treated with RQ1 RNase-free DNase (Promega), and cDNA made using Omniscript reverse transcriptase (Qiagen) and using random primers (Promega). Relative levels of SorCS2 gene expression were determined by RT-qPCR using Taqman® gene expression assays from Thermo Fisher Scientific [assay ID: Mm00473051_m1 and Mm00473072_m1] to detect the 5’ and 3’ regions of the major predicted transcript respectively, and Myo7a (assay ID: Rn00596450_m1). Reactions were multiplexed with a primer-limited eukaryotic 18S rRNA endogenous control, performed in triplicate for each animal and amplified on a SDS7500 real-time PCR System (Thermo Fisher Scientific). Relative quantification studies were performed using SDS1.2.1 software using the 2-ΔΔCT method (Thermo Fisher Scientific).
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