The same RNA samples used for microarray experiments were also used for qPCR. The cDNA synthesis was performed following the protocol as described.36 (link) RT-qPCR was performed using an ABI7500 thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA) with Bright Green PCR Master Mix (Sigma-Aldrich, St Louis, MO, USA). Melting cures and electrophoresis were performed to verify the specificity and identify of the PCR products. Citrus 5.8s rRNA gene was used for internal control as described71 and 23 genes were selected for validation. The relative quantification of gene expression level was determined by the comparative Ct method 2−ΔΔCt;,37 (link) where ΔΔCt=(Ct (target gene)—Ct (internal control))seedless—[Ct (target gene)—Ct (internal control)]seedy.
Abi 7500 thermal cycler
Manufactured by Thermo Fisher Scientific
Sourced in United States, Australia, China, Japan
The ABI 7500 thermal cycler is a real-time PCR system designed for quantitative gene expression analysis and genotyping. It features a block accommodating 96 standard sample tubes or microplates. The system utilizes a Peltier-based thermal block to precisely control sample temperatures during the PCR thermal cycling process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (34 mentions)
Trizol, by Thermo Fisher Scientific (7 mentions)
Primescript rt reagent kit, by Takara Bio (7 mentions)
Sybr green reagent, by Thermo Fisher Scientific (5 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
M mlv reverse transcriptase, by Promega (3 mentions)
Sybr premix ex taqtm kit, by Takara Bio (3 mentions)
Sybergreen, by Thermo Fisher Scientific (3 mentions)
Qscript cdna synthesis kit, by Quanta Biosciences (3 mentions)
Qiasymphony, by Qiagen (3 mentions)
Taqpath pcr covid 19 combo kit, by Thermo Fisher Scientific (3 mentions)
Xpert xpress sars cov 2, by Cepheid (3 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (2 mentions)
Hiscript 2 1st strand cdna synthesis kit, by Vazyme (2 mentions)
Sybr green pcr system, by Thermo Fisher Scientific (2 mentions)
Rt2 sybr green qpcr master mix, by Thermo Fisher Scientific (2 mentions)
Sybr premix ex taq, by Takara Bio (2 mentions)
Sybr premix ex taq tli rnaseh plus, by Takara Bio (2 mentions)
88 protocols using abi 7500 thermal cycler
1
RT-qPCR Validation of Microarray Data
2
Quantitative RT-PCR Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from LV tissues of rats (approximately 100 mg) using an Ultrapure RNA Kit (CWbio, Co., Ltd., Cat#CW0581) and reverse transcribed into cDNA using a PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Co., Ltd., Cat# RR047B) according to the manufacturer’s instructions. Real-time PCR reactions were carried out using the SYBR® Premix Ex Taq™ II (Tli RNaseH Plus), ROX plus (TaKaRa, Co., Ltd., Cat# RR82LR) in an ABI 7500 thermal cycler (Thermo Fisher Scientific, Inc.). The amplification was performed as follows: 1 cycle of 95°C for 30 s and 45 cycles of 95°C for 5 s and 60°C for 40 s. Primers and probes were verified as operating at similar efficiencies. The levels of GAPDH expression were measured in all samples to normalize gene expression for sample-to-sample differences in RNA input, RNA quality, and reverse transcription efficiency. The fold differences in mRNA expression levels between samples were calculated using the 2-ΔΔ Ct relative quantification method. The primer sequences (Invitrogen Co., Ltd., Beijing, China) are as follows (forward and reverse, 5′-3′):
3
Quantitative Analysis of Multidrug Resistance in HepG2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from the cells (HepG2 or HepG2/5-FU) with TRIzol® reagent (Sangon Biotech Co., Ltd.,), and cDNA synthesis was performed using the PrimeScript II 1st Strand cDNA Synthesis kit (Takara Bio, Inc.) according to the manufacturer's instructions. QPCR was performed with a SYBR® Green PCR system (Takara Bio, Inc.) in an ABI 7500 thermal cycler (Thermo Fisher Scientific, Inc.), and the thermocycling conditions were as follows: 94°C for 5 min; followed by 35 cycles of 94°C for 30 sec, 58°C for 30 sec and 72°C for 15 sec. The primers used were as follows: GAPDH forward, 5′-GCAGTGGCAAAGTGGAGATT-3′ and reverse, 5′-TGAAGTCGCAGGAGACAACC-3′; MDR1 forward, 5′-TCACTTCAGTTACCCATCTCG-3′ and reverse, 5′-CACCAATGATTTCCCGTAG-3′; P-gp forward, 5′-ACTTGCAAGGGGACCAGAGA-3′ and reverse, 5′-CCTTCAAGATCCATTCCGACC-3′; cyclin A forward, 5′-TCCATGTCAGTGCTGAGAGGC-3′ and reverse, 5′-GAAGGTCCATGAGACAAGGC-3′; cyclin B1 forward, 5′-ATGCAGCACCTGGCTAAGAA-3′ and reverse, 5′-TTACACCTTTGCCACAGCCT-3′; CDK2 forward, 5′-CTTTGCTGAGATGGTGACTCG-3′ and reverse, 5′-TCATCCAGGGGAGGTACAACT-3′; and caspase-3 forward, 5′-TGCATACTCCACAGCACCTG-3′ and reverse, 5′-TCTGTTGCCACCTTTCGGTT-3′. RT-qPCR for each sample was performed in duplicate. The fold-changes were calculated by relative quantification (2−ΔΔCq) (21 (link)). GAPDH was used as an internal control.
4
Quantitative RT-PCR of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from naive and BLP-tolerised BMMs at the indicated time periods using TRIzol (Invitrogen) and reverse-transcribed into cDNA using the ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). Amplification of cDNA was conducted using an ABI 7500 thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA). The target gene mRNA expression was analyzed with FastStart universal SYBR green master (Roche Life Science, Indianapolis, IN, USA) and normalised with the housekeeping gene β-actin. The gene-specific primers used in this study were listed in Table 1 .
5
Quantitative PCR Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA from RAW264.7 cells were extracted using TRIzol reagent according to the manufacturer’s instructions. First-strand cDNA synthesis was conducted using 1 μg of total RNA with HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). qPCR was performed on an ABI 7500 Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA) using All-in-One qPCR Mix (Genecopoeia, Guangzhou, China) under the following conditions: 95 °C for 10 min, 40 cycles for two steps (95 °C for 5 s and 60 °C for 1 min). The murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference gene. The 2−ΔΔCq method was used to calculate relative mRNA abundance [23 (link)]. The qPCR primers used in this study are listed in Table 1 .
6
Evaluation of Liver Cell Populations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For evaluation of the purity of liver cell populations and the expression of DEmRNAs, DElncRNAs and DEmiRNAs, qRT-PCR was performed. The genes for the cell markers (Alb, F4/80, α-SMA, Col1α1, Col3α1 and GFAP), DElncRNAs and their nearby genes located within the 10 kb upstream and downstream in the genome, and the metabolism- and inflammation-related DEmRNAs were selected for validation. 1 μg of total RNA was reversely transcribed to cDNA using HiScript Ⅲ First-strand cDNA Synthesis Kit (Vazyme, Munich, Germany) as recommended by the manufacturer. qRT-PCR was performed using All-in-OneTM qPCR Mix (GeneCopoeia) with an ABI 7500 Thermal Cycler (ThermoFisher Scientific, United States) according to the standard method. Specific primers for the selected DElncRNAs and DEmRNAs were obtained from TSINGKE (Xi’an, China) (Supplementary Table S1 ), and specific primers for miRNAs were purchased from GeneCopoeia (United States). The relative expression level was normalized to GAPDH for DEmRNAs and DElncRNAs or to U6 for DEmiRNAs. The 2−ΔΔCt algorithm was used to calculate the relative expression levels represented as relative fold-change (FC). All experiments were performed in triplicate.
7
RT-PCR and Q-PCR for Liver Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RT-PCR and quantitative real-time PCR (Q-PCR) were performed as previously described46 (link). Total RNA was extracted from liver tissues using TRIZOL reagent from Thermo Fisher Scientific (Waltham, MA, USA). The complementary DNA templates were obtained by reverse transcription in a 10 μL reaction containing 1 μg of total RNA, oligo (dT) primers, and a reverse transcription premix. Q-PCR was performed with the SYBR green PCR system in an ABI 7500 thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA). The SYBR green reagents were also purchased from Thermo Fisher Scientific. The following cycling conditions were used: 95 °C for 3 min followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 5 s, and extension at 72 °C for 10 s. The mRNA levels were normalized to the level of the β-actin mRNA, which was used as an internal control. The primers were: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), sense, 5′-CATTCAAGACCGGACAGAGG-3′, antisense, 5′-ACATACTGCACACCAGCATCACC-3′; HIPK2, sense, 5′-CCAGGC CTGCTTGCTCAG-3′, antisense, 5′-TGTACAGATGTGTGGGTGGC-3′. Finally, we determined the relative mRNA levels using the 2−ΔΔCt method, and GAPDH was used as an internal control.
8
Quantifying Gene Expression in A549 and HCT116 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A549 and HCT116 cells were treated with NaB (2 mM) at 37°C for 24 h, and total mRNA was extracted using TRIzol® reagent (Thermo Fisher Scientific Inc.). RNA concentration was determined using spectrophotometry and 500 ng RNA was used for cDNA synthesis. RT-qPCR was performed using the Takara SYBR® Premix Ex Taq™ system in order to quantify the expression levels of the target genes in an ABI 7500 thermal cycler (Thermo Fisher Scientific, Inc.). The thermocycling conditions for RT-qPCR were: Initial denaturation at 95°C for 30 sec, followed by 45 cycles at 95°C for 5 sec and 60°C for 30 sec; and a melt curve stage at 95°C for 15 sec, 60°C for 60 sec, 95°C for 30 sec and 60°C for 15 sec. GAPDH was selected as the housekeeping gene. After normalized to GAPDH gene, each target gene expression were calculated using the comparative threshold cycle (Cq) method (20 (link)). The ΔCq values were calculated according to the formula ΔCq=Cq (gene of interest)-Cq (GAPDH) in correlation analysis, and the 2−ΔΔCq was calculated according to the formula ΔΔCq=ΔCq (control group)-ΔCq (experimental group) for determination of relative. The sequences of the primers used in the RT-qPCR experiments are presented in Table I .
9
Quantification of Gene Expression by RT-PCR and Q-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The reverse transcription-polymerase chain reaction (RT-PCR) and the quantitative real-time PCR (Q-PCR) were performed as follows. Total RNA was extracted from lung tissues and cultured cells using TRIZOL reagent (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was obtained by reverse transcription in a 20-μL reaction containing 2 μg of total RNA, oligo (dT), and reverse transcription premix.
The quantitative real-time PCR (Q-PCR) reactions were performed with the SYBR green PCR system in an ABI 7500 thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA). The SYBR green reagents were also purchased from Thermo Fisher Scientific. The cycling conditions were as follows: 95°C for 3 min; followed by 40 cycles involving denaturing at 95°C for 10 s, annealing at 60°C for 5 s, and extension at 72°C for 10 s. Expression of mRNAs was normalized by the mRNA levels of GAPDH, which was used as an internal control. We analyzed the relative levels of mRNAs using the 2−ΔΔCt method, and GAPDH was used as the internal control. The primer sequence was: HIPK1: forward, 5′-TCCCCATACTACGAGAAGGGT-3′; reverse, 5′-ATGTCCCCACCCCTAGTACC-3′; GAPDH: forward, 5′-CATTCAAGACCGGACAGAGG-3′; reverse, 5′-ACATACTGCAC ACCAGCATCACC -3′.
The quantitative real-time PCR (Q-PCR) reactions were performed with the SYBR green PCR system in an ABI 7500 thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA). The SYBR green reagents were also purchased from Thermo Fisher Scientific. The cycling conditions were as follows: 95°C for 3 min; followed by 40 cycles involving denaturing at 95°C for 10 s, annealing at 60°C for 5 s, and extension at 72°C for 10 s. Expression of mRNAs was normalized by the mRNA levels of GAPDH, which was used as an internal control. We analyzed the relative levels of mRNAs using the 2−ΔΔCt method, and GAPDH was used as the internal control. The primer sequence was: HIPK1: forward, 5′-TCCCCATACTACGAGAAGGGT-3′; reverse, 5′-ATGTCCCCACCCCTAGTACC-3′; GAPDH: forward, 5′-CATTCAAGACCGGACAGAGG-3′; reverse, 5′-ACATACTGCAC ACCAGCATCACC -3′.
10
Profiling Circular RNA Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA extracted from PBMCs and cell lines was subjected to RT-qPCR validation. Specific divergent primers spanning the back-splice junction sites of the circRNAs were designed. To detect the expression levels of the selected circRNAs, cDNA was synthesized using the PrimeScript™ RT reagent kit (Takara Biotechnology Co., Ltd.) by incubating at 37˚C for 15 min and then 85˚C for 5 sec to terminate the reaction, with a final volume of 20 µl. TB Green Premix Ex Taq™ II (Takara Biotechnology Co., Ltd.) was used for qPCR analyses on ABI 7500 thermal cycler according to the manual instruction (Thermo Fisher Scientific, Inc.). After predenaturing at 95˚C for 2 min, the reaction system were submitted to 94˚C for 15 sec and 60˚C, 45 sec, for 40 cycles. The internal control gene GAPDH was measured at the same time. The expression of different circRNAs was compared using the 2-ΔΔCq method (41 (link)). The primers (synthesized by Sangon Biotech) used in the present study are listed in Table I .
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!