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Abi 7900ht cycler

Manufactured by Thermo Fisher Scientific
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The ABI 7900HT cycler is a real-time PCR system designed for high-throughput nucleic acid analysis. It features a 384-well sample capacity and supports various chemistries and experimental designs. The system provides accurate and reliable data collection and analysis for a range of applications.

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Lab products found in correlation

Sybr green master mix, by Thermo Fisher Scientific (3 mentions) High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions) Rneasy micro kit, by Qiagen (2 mentions) Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions) Tetro reverse transcription kit, by Meridian Bioscience (1 mentions) Sensifast sybr hi rox mix, by Meridian Bioscience (1 mentions) High pure rna isolation kit, by Roche (1 mentions) Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions) Mir x mirna first strand synthesis kit, by Takara Bio (1 mentions) Sybr advantage qpcr premix, by Takara Bio (1 mentions) High capacity complementary dna reverse transcription kit, by Thermo Fisher Scientific (1 mentions) Taqman fast universal pcr master mix, by Thermo Fisher Scientific (1 mentions) Taqman probe, by Thermo Fisher Scientific (1 mentions)

6 protocols using abi 7900ht cycler

1

RNA Isolation and qPCR Analysis

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Cells grown to confluence in 6-well plates were washed once with PBS. Cells were harvested and total RNA was isolated with the High Pure RNA Isolation Kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. RNA concentrations were measured with a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). 1 μg of total RNA was reversely transcribed into cDNA with the Tetro reverse transcription kit (Bioline, Luckenwalde, Germany). Quantitative real-time PCR was carried out using SensiFAST SYBR Hi-ROX Mix (Bioline) on an ABI 7900HT cycler (Applied Biosystems, Darmstadt, Germany). HPRT1 was used as housekeeper gene. For analysis of relative changes, data was analyzed according to the ΔΔCT method.
Hofmann N., Galetskiy D., Rauch D., Wittmann T., Marquardt A., Griese M, & Zarbock R. (2016). Analysis of the Proteolytic Processing of ABCA3: Identification of Cleavage Site and Involved Proteases. PLoS ONE, 11(3), e0152594.
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2

Quantitative RT-PCR for KLF5 Expression

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Extracted RNA was treated with DNase 1, and complimentary DNA (cDNA) was reverse transcribed from RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). TaqMan Gene Expression Assays were used for KLF5 and GAPDH (details available from authors). The absolute expression of KLF5 was quantified using qRT-PCR using the ABI 7900HT cycler (Applied Biosystems), and the critical threshold was manually set at 0.2. Relative expression was calculated using the ΔΔCT method described by Livak and Schmittgen75 (link), with GAPDH as an endogenous control.
Cheng T.H., Thompson D.J., O’Mara T.A., Painter J.N., Glubb D.M., Flach S., Lewis A., French J.D., Freeman-Mills L., Church D., Gorman M., Martin L., Hodgson S., Webb P.M., Attia J., Holliday E.G., McEvoy M., Scott R.J., Henders A.K., Martin N.G., Montgomery G.W., Nyholt D.R., Ahmed S., Healey C.S., Shah M., Dennis J., Fasching P.A., Beckmann M.W., Hein A., Ekici A.B., Hall P., Czene K., Darabi H., Li J., Dörk T., Dürst M., Hillemanns P., Runnebaum I., Amant F., Schrauwen S., Zhao H., Lambrechts D., Depreeuw J., Dowdy S.C., Goode E.L., Fridley B.L., Winham S.J., Njølstad T.S., Salvesen H.B., Trovik J., Werner H.M., Ashton K., Otton G., Proietto T., Liu T., Mints M., Tham E., Consortium C., Jun Li M., Yip S.H., Wang J., Bolla M.K., Michailidou K., Wang Q., Tyrer J.P., Dunlop M., Houlston R., Palles C., Hopper J.L., Peto J., Swerdlow A.J., Burwinkel B., Brenner H., Meindl A., Brauch H., Lindblom A., Chang-Claude J., Couch F.J., Giles G.G., Kristensen V.N., Cox A., Cunningham J.M., Pharoah P.D., Dunning A.M., Edwards S.L., Easton D.F., Tomlinson I, & Spurdle A.B. (2016). Five endometrial cancer risk loci identified through genome-wide association analysis. Nature genetics, 48(6), 667-674.
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3

Quantitative mRNA Expression Analysis

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mRNA was isolated from human and murine liver tissues using Trizol and following manufacturer’s manual instructions (Invitrogen, Carlsbad, CA, USA). mRNA from sorted cells was harvested by using the QIAGEN RNeasy MICROKit (QIAGEN GmbH, Hilden, Germany). mRNA levels were evaluated by quantitative real time PCR on an ABI 7900HT cycler (Applied Biosystems) using Taqman gene expression assays (Life technologies). Individual gene expression was normalized to 18 s or GAPDH. Relative expression was calculated using the comparative Ct method (2−ΔΔCt).
Graupera I., Coll M., Pose E., Elia C., Piano S., Solà E., Blaya D., Huelin P., Solé C., Moreira R., de Prada G., Fabrellas N., Juanola A., Morales-Ruiz M., Sancho-Bru P., Villanueva C, & Ginès P. (2017). Adipocyte Fatty-Acid Binding Protein is Overexpressed in Cirrhosis and Correlates with Clinical Outcomes. Scientific Reports, 7, 1829.
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4

Quantitative RT-PCR for KLF5 Expression

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Extracted RNA was treated with DNase 1, and complimentary DNA (cDNA) was reverse transcribed from RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). TaqMan Gene Expression Assays were used for KLF5 and GAPDH (details available from authors). The absolute expression of KLF5 was quantified using qRT-PCR using the ABI 7900HT cycler (Applied Biosystems), and the critical threshold was manually set at 0.2. Relative expression was calculated using the ΔΔCT method described by Livak and Schmittgen75 (link), with GAPDH as an endogenous control.
Cheng T.H., Thompson D.J., O’Mara T.A., Painter J.N., Glubb D.M., Flach S., Lewis A., French J.D., Freeman-Mills L., Church D., Gorman M., Martin L., Hodgson S., Webb P.M., Attia J., Holliday E.G., McEvoy M., Scott R.J., Henders A.K., Martin N.G., Montgomery G.W., Nyholt D.R., Ahmed S., Healey C.S., Shah M., Dennis J., Fasching P.A., Beckmann M.W., Hein A., Ekici A.B., Hall P., Czene K., Darabi H., Li J., Dörk T., Dürst M., Hillemanns P., Runnebaum I., Amant F., Schrauwen S., Zhao H., Lambrechts D., Depreeuw J., Dowdy S.C., Goode E.L., Fridley B.L., Winham S.J., Njølstad T.S., Salvesen H.B., Trovik J., Werner H.M., Ashton K., Otton G., Proietto T., Liu T., Mints M., Tham E., Consortium C., Jun Li M., Yip S.H., Wang J., Bolla M.K., Michailidou K., Wang Q., Tyrer J.P., Dunlop M., Houlston R., Palles C., Hopper J.L., Peto J., Swerdlow A.J., Burwinkel B., Brenner H., Meindl A., Brauch H., Lindblom A., Chang-Claude J., Couch F.J., Giles G.G., Kristensen V.N., Cox A., Cunningham J.M., Pharoah P.D., Dunning A.M., Edwards S.L., Easton D.F., Tomlinson I, & Spurdle A.B. (2016). Five endometrial cancer risk loci identified through genome-wide association analysis. Nature genetics, 48(6), 667-674.
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5

Quantitative Analysis of miRNA and mRNA

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Expression of mature miRNAs was assessed by reverse transcription using Mir-X miRNA First-strand Synthesis Kit (Takara, Dalian, China) according to the manufacturer’s instructions and qPCR reaction was performed by using SYBR® Advantage qPCR premix (Takara). mRNA levels were determined by quantitative real time PCR on an ABI 7900HT cycler (Applied Biosystems) using SYBR green master mix (Life technologies). Individual gene and miRNA expression was normalized to GAPDH or U6 expression, respectively. Relative expression was calculated using the comparative Ct method (2−ΔΔCt). Gene specific primers were produced by Integrated DNA Technologies (Leuven, Belgium). Primer sequences used are listed in Supplementary Table 3.
Coll M., Taghdouini A.E., Perea L., Mannaerts I., Vila-Casadesús M., Blaya D., Rodrigo-Torres D., Affò S., Morales-Ibanez O., Graupera I., Lozano J.J., Najimi M., Sokal E., Lambrecht J., Ginès P., van Grunsven L.A, & Sancho-Bru P. (2015). Integrative miRNA and Gene Expression Profiling Analysis of Human Quiescent Hepatic Stellate Cells. Scientific Reports, 5, 11549.
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6

Gene Expression Quantification Protocol

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RNA from sorted cells was obtained using the RNeasy Micro Kit (QIAGEN). Total RNA was retro-transcribed using a High Capacity Complementary DNA Reverse-Transcription kit (Thermo Fisher Scientific). Gene expression was determined by quantitative real time PCR on an ABI 7900HT cycler (Applied Biosystems) using SYBR green master mix (Thermo Fisher Scientific) or Taqman FAST Universal PCR Master Mix (Thermo Fisher Scientific). Individual gene expression was normalized to GAPDH. Relative expression was calculated using the comparative Ct method (2 ÀDDCt ). Gene specific primers were produced by Integrated DNA Technologies (Leuven, Belgium) and TaqMan probe by Thermo Fisher Scientific. Primers sequences are listed in Table S4.
Coll M., Perea L., Boon R., Leite S.B., Vallverdú J., Mannaerts I., Smout A., El Taghdouini A., Blaya D., Rodrigo-Torres D., Graupera I., Aguilar-Bravo B., Chesne C., Najimi M., Sokal E., Lozano J.J., van Grunsven L.A., Verfaillie C.M, & Sancho-Bru P. (2018). Generation of Hepatic Stellate Cells from Human Pluripotent Stem Cells Enables In Vitro Modeling of Liver Fibrosis. Cell stem cell, 23(1).
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