7500 sequence detection system apparatus

Manufactured by Thermo Fisher Scientific

Sourced in United States

The 7500 Sequence Detection System is a real-time PCR instrument designed for gene expression analysis, SNP genotyping, and other nucleic acid detection applications. It features a 96-well format, supports multiple fluorescent dye detection, and provides accurate and reproducible results.

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Lab products found in correlation

Taqman master mix, by Thermo Fisher Scientific (6 mentions) Trizol reagent, by Thermo Fisher Scientific (5 mentions) High capacity kit, by Thermo Fisher Scientific (3 mentions) High capacity cdna kit, by Thermo Fisher Scientific (2 mentions) Abi 7500 sds software, by Thermo Fisher Scientific (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Omniscript kit, by Qiagen (1 mentions) Sybr green 1 kit, by Takara Bio (1 mentions)

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7500 Sequence Detection System (208 citations) 7500 system (124 citations) Sequence Detection Systems (5 citations) 7500 apparatus (5 citations)

7 protocols using 7500 sequence detection system apparatus

Quantitative RT-PCR for Calcitonin Receptor-Like Receptor Gene Expression

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Total cellular RNA was extracted using Trizol^¯ Reagent (Invitrogen, USA),
and RNA was reverse transcribed to single-stranded cDNA using a High Capacity Kit
(Applied Biosystems, USA) according to the manufacturer's protocol. For quantitative
analysis of the genes of interest [pre-pro-AM (Rn 00562327_m1), CRLR (Rn
00562334_m1), RAMP1 (Rn 01427056_m1), RAMP2 (Rn 00824652_m1), and RAMP3 (Rn
00571815_m1)], a commercially available TaqMan Assay-on-Demand System that consists
of a kit of oligonucleotides and probes was used (Applied Biosystems). Reverse
transcription was performed using 1 µg total RNA for each sample in 20 µL of the
total reaction mixture. The cDNA obtained was diluted 1:10, and 4.5 µL was used for
each 10 µL of the qRT-PCR mixture using the TaqMan Master Mix (Applied Biosystems).
Reactions were carried out in duplicate and analyzed with 7500 Sequence Detection
System apparatus (Applied Biosystems). Data were analyzed using the ABI-7500 SDS
software (Applied Biosystems). Total RNA absorbed was normalized on the basis of the
Ct value for the GAPDH gene (Rn 01775763_m1). The variation in expression among
samples was calculated by the 2^−ΔΔCt method, and the mean delta Ct value
for a group of six samples from the control was used for calibration (17 (link)).

Leite L.N., Gonzaga N.A., Tirapelli D.P., Tirapelli L.F, & Tirapelli C.R. (2014). Pharmacological characterization of the relaxant effect induced by adrenomedullin in rat cavernosal smooth muscle. Brazilian Journal of Medical and Biological Research, 47(10), 876-885.

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Quantitative Analysis of DNA Methyltransferases and TET Enzymes in Bladder Tissues

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Total RNA was isolated from bladder tissues with the use of the RNeasy Mini kit (Qiagen, Inc.). The cDNA was then synthesized from 1 µg of total RNA using the Omniscript kit (Qiagen, Inc.) by random decamer primers (Applied Biosystems/Ambion; Thermo Fisher Scientific, Inc.). The reaction was incubated at 37°C for 60 min, inactivated by heating at 95°C for 5 min, and stored at -20°C. An SYBR-Green I kit (Takara Biotechnology, Co., Ltd.) was used and all the primers of rat DNA methyltransferase (DNMT1, 3a, and 3b) and TET enzymes (TET1, TET2, and TET3) were listed in Table I. RT-qPCR was performed in a 7500 Sequence Detection System apparatus (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the following thermocycling conditions: Initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The relative expression levels of each targeted gene were normalized by subtracting the corresponding β-actin threshold cycle (Cq) values using the ΔΔCq comparative method (36 (link)). Six samples for each group were used and run in triplicate.

Chuang S.M., Lu J.H., Lin K.L., Long C.Y., Lee Y.C., Hsiao H.P., Tsai C.C., Wu W.J., Yang H.J, & Juan Y.S. (2019). Epigenetic regulation of COX-2 expression by DNA hypomethylation via NF-κB activation in ketamine-induced ulcerative cystitis. International Journal of Molecular Medicine, 44(3), 797-812.

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Quantifying miRNA-21 Expression in Tissue

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The expression profile of the miRNA-21 was analyzed in blood and the cavernous tissue samples from each animal. Total cellular RNA was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA) and RNA was reverse transcribed to single-stranded cDNA, using a High Capacity Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol. For quantitative analysis of the miRNA-21 (002493), we used the commercially available system TaqMan Assay-ondemand (Applied Biosystems). Reverse transcription was performed using 5ng total RNA for each sample in 7,5µL of the total reaction mixture. The cDNA obtained was diluted 1:4 and 4.5µL was used for each 10µL of the quantitative real-time polymerase chain reaction mixture using the TaqMan Master Mix (Applied Biosystems). All reactions were carried out in duplicate and analyzed with the 7500 Sequence Detection System apparatus (Applied Biosystems). Data were analyzed using the ABI-7500 SDS software. The total RNA absorbed was normalized on the basis of the Ct value for U6 (000391). The variation in expression among samples was calculated by the 2-∆∆Ct method, with the mean ∆Ct value for a group of 6 samples from control rats being used as a calibrator.

Meirelles R.J., Lizarte FS N.e.t.o., Cirino M.L., Novais P.C., Gula I.S., da Silva J.P., Tazzima M.D., Fazan V.P., Durand M.T., Tirapelli D.P., de Carvalho C.A., Schimming B.C., Molina C.A., Tucci S J.u.n.i.o.r, & Tirapelli L.F. (2020). Morphological and molecular analysis of apoptosis in the corpus cavernosum of rats submitted to a chronic alcoholism model. Acta Cirúrgica Brasileira, 35(3), e202000305.

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Quantitative Analysis of Apoptosis Genes

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Total cellular RNA was extracted with Trizol Reagent (Applied Biosystems, USA) according to the manufacturer’s instructions. In preparation of Real-Time Polymerase Chain Reaction (PCR), reverse transcription of RNA samples was performed using the High-Capacity cDNA kit (Applied Biosystems, USA). For quantitative analysis of the genes Caspase-3 and -9 (Assay ID Hs00234385_m1; Rn00581212_m1, respectively), we used the commercially available system TaqMan Assay-on-demand (Applied Biosystems). The cDNA was amplified with quantitative Real Time Polymerase Chain Reaction (q-PCR) using TaqMan Master Mix (Applied Biosystems, USA) for gene reaction. The total RNA absorbed was normalized on the basis of the Ct value for β-actin (ACT-β) gene (Rn00667869-m1). All reactions were carried out in duplicate and analyzed with the 7500 Sequence Detection System apparatus (Applied Biosystems, USA). Data were analyzed using the ABI-7500 SDS software. Relative quantification of the examined genes was calculated with a calibrator by using the 2^-method.

Schimming B.C., Cirino M.L., Lizarte FS N.e.t.o., Novais P.C., de Carvalho C.A., Tirapelli D.P., Molina C.A., Tirapelli L.F, & Tucci S J.u.n.i.o.r. (2020). Chronic alcoholism associated with diabetes induced apoptosis in the corpus cavernosum of rats. Acta Cirúrgica Brasileira, 35(8), e202000805.

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Quantifying miRNA-155 and miRNA-199 Expression

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The expression profile of the miRNAs-155 and -199 were analyzed in blood and cavernous tissue samples from each animal. Total cellular RNA was extracted using Trizol Reagent (Invitrogen, USA) and RNA was reverse transcribed to single-stranded cDNA, using a High Capacity Kit (Applied Biosystems, USA) according to the manufacturer's protocol. For quantitative analysis of the miRNAs-155 and -199, we used the commercially available system TaqMan Assay-ondemand (Applied Biosystems). Reverse transcription was performed using 5 ng total RNA for each sample in 7.5 µL of the total reaction mixture. The cDNA obtained was diluted 1:4 and 4.5 µL was used for each 10 µL of the quantitative real-time polymerase chain reaction mixture using the TaqMan Master Mix (Applied Biosystems). All reactions were carried out in duplicate and analyzed with the 7500 Sequence Detection System apparatus (Applied Biosystems). Data were analyzed using the ABI-7500 SDS software. The total RNA absorbed was normalized based on the Ct value for U6 (000391). The variation in expression among samples was calculated by the 2-ΔΔCt method, with the mean ΔCt value for a group of 6 samples from control rats used as a calibrator.

Gonçalves F.Z., Lizarte FS N.e.t.o., Novais P.C., Gattas D., Lourenço L.G., de Carvalho C.A., Tirapelli D.P., Molina C.A., Tirapelli L.F, & Tucci S J.r. (2018). Expression profile of endothelin receptors (ETA and ETB) and microRNAs-155 and -199 in the corpus cavernosum of rats submitted to chronic alcoholism and diabetes mellitus. Brazilian Journal of Medical and Biological Research, 51(3), e6329.

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Quantitative Real-Time PCR for miR-143

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Total RNA was extracted using a Trizol reagent (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions. In preparation for the real-time Polymerase Chain Reaction (PCR), reverse transcription of RNA samples was performed using the High-Capacity cDNA kit (Applied Biosystems). The cDNA was amplified with Quantitative real-time Polymerase Chain Reaction (Q-PCR) using TaqMan Master Mix (Life Technologies, Carlsbad, CA, USA) to respond to microRNAs. The specific probes and assay ID for miR-143 was 000466 from qPCR Taqman™ (Life Technologies). The U6 gene was used as an endogenous control (housekeeping) for the reaction of the microRNA. The PCR conditions included pre-heating at 50 °C for two minutes, denaturation at 95 °C for ten minutes, and 50 cycles of amplification and quantification (15 s at 95 °C and one minute at 60 °C). The 7500 Sequence Detection System apparatus (Applied Biosystems) duplicated and analyzed all reactions. The data were analyzed using ABI-7500 SDS software. Dissociation curves were performed (melting curves) after amplification by RQ-PCR. The samples that showed dissociation curves with different temperatures or more than one point of dissociation in the same sample were discarded and repeated for miRNA analysis (n = 6 per group).

Diniz A.M., Gualberto I.J., Lima L.A., Cirino M.L., Murakami R.K., Ishikiriama B.L., Ruano R., da Silva L.F., Tirapelli D, & Sbragia L. (2023). miRNA-143 expression is associated with inflammation and time of exposure to amniotic fluid in experimental gastroschisis. Clinics, 78, 100311.

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Quantitative Analysis of Gene Expression

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For the analysis of gene expression, 10 animals were used per group. A fragment was obtained from each animal and 1 ml of blood was collected at a single moment in the four study groups.
Total RNA was extracted with Trizol reagent (Applied Biossystems, USA) according to the manufacture's instructions. To verify the integrity of the RNA obtained, each sample was subjected to electrophoresis on agarose gel 1% RNA and put through the spectrophotometer that provides the RNA concentration in a sample of 1 to 2 μl. In addition to the concentration, this device provides us with values relating to the integrity of the samples (260/280 ratio). The ideal range to be obtained is 1.7 to 1.9.
To prepare the real-time polymerase chain reaction (PCR), reverse transcription of RNA samples was performed using the High-Capacity cDNA kit (Applied Biossystems, USA). The cDNA was amplified with quantitative real-time PCR using the TaqMan Master Mix (Applied Biosystems) for miRNA reaction.
Endogenous control for the reaction of the miRNA and the gene, was through the use of U6 and GAPDH, respectively. All reactions were carried out in duplicate and analyzed with the 7500 Sequence Detection System apparatus (Applied Biosystems).

Silva C.I., Novais P.C., Rodrigues A.R., Carvalho C.A., Colli B.O., Carlotti CG J.r., Tirapelli L.F, & Tirapelli D.P. (2017). Expression of NMDA receptor and microRNA-219 in rats submitted to cerebral ischemia associated with alcoholism. Arquivos de neuro-psiquiatria, 75(1).

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