For determination of the copy number of the transgene donor cassette in EGFP transgenic mice and Tbx3-3xFlag-2A-EGFP knock-in mice, real-time PCR was performed in the E17.5 and E15.5 embryonic tail genomes. qPCR was conducted in duplicate with 20 ng of DNA in a 20 µL reaction mixture using the THUNDERBIRD SYBR qPCR Mix (TOYOBO) and the Applied Biosystems 7500 Real-Time system (Applied Biosystems). R26R-H2B-EGFP heterozygous and homozygous knock-in mouse tail genomes were used as references for copy number. DNA amount was calibrated using the PCR product of the CriMGET-OT1 region on chromosome 8 as an internal control. Copy number was defined as the value relative to the R26R-H2B-EGFP heterozygous knock-in sample.
Applied biosystems 7500 real time system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Applied Biosystems 7500 Real-Time system is a powerful and versatile real-time PCR instrument designed for a wide range of applications. It features a compact design, intuitive software, and reliable performance to support your research needs.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (11 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (6 mentions)
Rnaiso plus, by Takara Bio (3 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (3 mentions)
Thunderbird sybr qpcr mix, by Toyobo (2 mentions)
Sybr premix ex taq reagent, by Takara Bio (2 mentions)
Revetra ace qpcr rt master mix, by Toyobo (2 mentions)
Rnaiso plus reagent, by Takara Bio (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Moloney murine leukemia virus system, by Thermo Fisher Scientific (2 mentions)
Quantstudio 6 flex real time pcr system, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Takara Bio (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Isogen, by Nippon Gene (1 mentions)
Revertra ace qpcr rt master mix, by Toyobo (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (1 mentions)
Lightcycler 480 instrument, by Roche (1 mentions)
26 protocols using applied biosystems 7500 real time system
1
Determination of Transgene Copy Number
2
Quantifying tdTomato Expression in Transgenic Mice
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For determination of the tdTomato gene expression in F0 4-week-old Hipp11CAG-tdTomato knock-in mouse, real-time PCR was performed on RNA from the brain, heart, lung, liver, kidney, spleen, intestine, and skeletal muscle tissue. RNA from each tissue was extracted using ISOGEN (NIPPON GENE), in accordance with the manufacturer’s protocol. Samples were treated with DNase I (Takara) at 37 °C for 20 min, after which RNAs were purified with RNeasy Mini kit (Qiagen) for clean-up, as per the manufacturer’s protocol. cDNAs were synthesized by using ReverTra Ace® qPCR RT Master Mix (TOYOBO) and applied to a qPCR reaction mixture (20 µL) of the THUNDERBIRD SYBR qPCR Mix (TOYOBO). The reactions were performed in duplicate for each sample using the Applied Biosystems 7500 Real-Time system (Applied Biosystems). The following primer pairs were used: tdTomato_qPCR_Fw: 5′-atcgtggaacagtacgagcg-3′, tdTomato_qPCR_Rev: 5′-tgaactctttgatgacggcca-3′, and G3pdh_qPCR_Fw: 5′-aggtcggtgtgaacggatttg-3′, and G3pdh_qPCR_Rev: 5′-tgtagaccatgtagttgaggtca-3′.
3
Electrical Stimulation and Drug Effects on mRNA Expression
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The mRNA expression levels of integrin β1, TGF-β1, and COL I and III in cells were evaluated by RT-qPCR after electrical stimulation or/and drug incubation. The primers used for amplification were purchased from Beijing SBS Genetech Co., Ltd. Total RNA from FVWFs was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), following the manufacturer's protocol. RNA (2 µg) was reverse transcribed to cDNA (n=3; temperature protocol: 25°C for 5 min, 42°C for 30 min and 85°C for 5 min) using a Revert Aid First Strand cDNA Synthesis kit (cat. no. k1622; Thermo Fisher Scientific, Inc.) and reaction mixture aliquots (1 µl) were used as templates for PCR. Primer sequences for TIMP-1, MMP-2, MMP-9, COL I and III, and GAPDH were purchased from SBS Genetech Co., Ltd. (Table I ). qPCR was performed using SYBR® Premix Ex Taq™ reagent (cat. no. DRR041; Takara Bio, Inc.) and an Applied Biosystems 7500 Real-Time system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were: 40 cycles of initial denaturation, 95°C for 5 min; denaturation, 95°C for 10 sec; anneal, 55°C for 20 sec; and extension, 72°C for 20 sec. Normalized quantification cycle (Cq) values were used for comparison (21 (link)). Each sample was analyzed in triplicate.
4
Quantifying Apoptosis-Related Genes in PLFs
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The mRNA expression levels of Nr4a1, Bcl2, Bax, and Caspase 3 in PLFs were evaluated by RT-qPCR. The primers used for amplification were purchased from Beijing SBS Genetech Co., Ltd. (Beijing, China) (Table 1 ). The total RNA was extracted from PLFs using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the manufacturer's protocol. cDNA was prepared by the reverse transcription of total RNA(100 ng) using a Revert Aid First Strand cDNA Synthesis kit (catalog No. k1622; Thermo Fisher Scientific, Inc. USA) and reaction mixture aliquots (1 μL) were used as templates for PCR. qPCR was performed on an Applied Biosystems 7500 Real-Time system (Applied Biosystems, Thermo Fisher Scientific, Inc. USA) using SYBR® Premix Ex Taq™ reagent (catalog No. DRR041; TakaRa Bio, Inc., Otsu, Japan). Target gene mRNA expression levels were normalized to the expression of the housekeeping gene GAPDH for quantification. Each sample was analyzed in triplicate to ensure accuracy.
5
Quantitative Analysis of mRNA and miRNA Levels
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Total RNAs, including miRNAs, were extracted from cells using RNA iso plus reagent (Takara, Otsu, Japan). For analysis of mRNA expression levels, 1 μg of isolated RNA was reverse-transcribed using ReveTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). C9orf3, CLCN3, KCNK2, FOXP2, PKIA, SEC24A, TRIL, and ZDHHC17 mRNA levels were measured using SYBR Green Master Mix and Applied Biosystems 7500 Real-time System (Applied Biosystems, Foster City, CA, USA). The sequences of primer sets are provided in Table S1 . mRNA levels were measured by the comparative ΔΔCt method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as a control and expressed as values relative to the indicated control sample. For analysis of miRNA expression levels, isolated RNAs were cleaned using miRNeasy mini kit (Qiagen, Hilden, Germany) and reverse-transcribed using miRNURYLNA Universal RT kit according to the manufacturer’s protocol. Locked nucleic acid (LNA) PCR primer sets (Exiqon) targeting hsa-miR-23b-3p (Product No. 204790), hsa-miR-27b-3p (Product No. 205915), hsa-miR-24-3p (Product No. 204260), and hsa-miR-10a-5p (Product No. 204788) were used to detect miRNA expression. miRNA levels were measured by the comparative ΔΔCt method using RNU48 as a control and expressed as values relative to the indicated control sample.
6
Analyzing EMT-related Genes in Colon Cancer
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Total RNA was extracted from cells using RNA iso plus reagent (Takara, Otsu, Japan). The isolated RNA (1 μg) was then reverse-transcribed using ReveTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). CDH1, ZEB1, SNAIL1, VIM, and ZNF350 mRNA levels were measured using SYBR Green Master Mix with an Applied Biosystems 7500 Real-time System (Applied Biosystems, Foster City, CA, USA). The sequences of the primer sets are listed in Supplementary Table 1 . The target mRNA levels were calculated uding the comparative ΔΔCt method with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA as an endogenous quantitative control. The expression of each gene is presented as the relative change compared to that in the indicated control sample.
Further, a TissueScan qPCR Array (HCRT103), which includes cDNAs from the paired normal and tumor tissues of 22 patients with colon adenocarcinoma (5 cases in stage I, 7 cases in stage II, 8 cases in stage III, and 2 cases in stage IV), was obtained from OriGene Technologies. Using this array, ZNF350 mRNA levels in human colon adenocarcinoma were measured by qPCR and normalized to those of GAPDH mRNA.
Further, a TissueScan qPCR Array (HCRT103), which includes cDNAs from the paired normal and tumor tissues of 22 patients with colon adenocarcinoma (5 cases in stage I, 7 cases in stage II, 8 cases in stage III, and 2 cases in stage IV), was obtained from OriGene Technologies. Using this array, ZNF350 mRNA levels in human colon adenocarcinoma were measured by qPCR and normalized to those of GAPDH mRNA.
7
Quantitative Analysis of DlCDPs
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Total RNA was isolated from germination buds and dormant buds using RNA prep Pure Plant Kit (Tiangen, Beijing, China), and reverse transcribed into cDNA using PrimeScript™ RT reagent Kit (Perfect Real Time, Takara, Japan). Quantitative real-time PCR analysis of 8 selected DlCDPs was performed on a LightCycler480 instrument (Roche) using Taq Pro Universal SYBR qPCR Master Mix SYBR Green premix Ex Taq Kit (TaKaRa, Dalian, China) on an Applied Biosystems 7500 Real-Time System (Applied Biosystems, Foster City, CA, USA). The q-PCR amplification program was: 95°C pre-denaturation for 5 seconds, 95°C denaturation for 30 seconds, 60°C annealing for 30 seconds; 72°C extension for 30 seconds, 40 cycles. GAPDH gene was used as an internal reference gene (Liu et al., 2014 (link)), and the relative expression of each gene was calculated using the 2-ΔΔCT method. The primers used in this study are shown in Table S5
8
Quantifying TRA2B1, TRA2B4, and CDKNA1 Expression
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Total RNAs were extracted from cells using TRIzol reagent (Life Technologies). One microgram of isolated RNA was reverse-transcribed using a PrimeScript RT Reagent Kit (Takara, Otsu, Japan). TRA2β1, TRA2β4 and CDKNA1 mRNA levels were measured using SYBR Green Master Mix and Applied Biosystems 7500 Real-time System (Applied Biosystems, Foster City, CA, USA). The sequences of primer sets are provided in Supplementary Table S1 . TissueScan Tissue qPCR Arrays (HCRT103) including cDNAs from paired normal and tumor tissues in 24 patients with adenocarcinomas of the colon were obtained from OriGene Technologies (Rockville, MD, USA), and TRA2β4 levels in normal and tumor tissues were determined by qPCR. TRA2β4 levels were measured by the comparative ΔΔCt method using ACTB mRNA as a control and expressed as values relative to the normal samples.
9
Quantification of Gene Expression in E. coli
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Overnight cultures of WT and ΔrpoS were statically subcultured in DMEM at 28°C for 12 h, respectively. RNA samples were extracted with the RNA isolation kit (Tiangen) as previously described [13 (link)] and incubated with DNase I (Promega) for 30 min at 37°C to remove genomic DNA. RNA concentrations were measured with NanoDrop and 1 μg of each sample was used for reverse transcription with PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa). The qRT-PCR was conducted on the Applied Biosystems 7500 real-time system (Applied Biosystems, Foster City, CA) in triplicate. The comparative CT (2-ΔΔCT) method was used to quantify the relative qualities of each transcript, and the housekeeping gyrB gene was used as an internal control. All the primers used are listed in S7 Table .
10
Quantitative RT-PCR Analysis
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Equal amounts of RNA (1 μg) were used to generate complementary DNA (Toyobo, Tsuruga, Japan) using random primers. Three independent qRT-PCR experiments were performed, and each was run in triplicate. The specific primer pairs that were used are shown in S2 Table . Reactions were run on an Applied Biosystems 7500 Real Time System (Applied Biosystems), and transcript levels were normalized to 16 sRNA in each sample using the ΔΔCT method.
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