Prism 7900ht fast real time pcr system

Manufactured by Thermo Fisher Scientific

Sourced in United States

The PRISM 7900HT Fast Real-Time PCR System is a laboratory instrument designed for real-time polymerase chain reaction (PCR) analysis. It is capable of performing rapid, sensitive, and accurate quantification of DNA and RNA targets.

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22 protocols using prism 7900ht fast real time pcr system

Genotyping Cardiovascular Disease SNPs

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Genomic DNA was extracted from peripheral blood using standard methods (DNA Blood Mini kit, QIAGEN, Hilden, Germany). All polymorphisms were genotyped using specific TaqMan assays on a Prism 7900HT Fast Real-Time PCR system following the supplier’s instructions (ThermoFisher Scientific, Foster City, CA, USA).
The polymorphic sites were selected by a prior review of the NCBI data base, and included polymorphisms with a minor allele frequency (MAF) >5%, previously demonstrated to be associated with cardiovascular diseases and with vascular calcification.

Cazarín-Santos B.G., Pérez-Hernández N., Posadas-Sánchez R., Vargas-Alarcón G., Pérez-Méndez Ó., Rodríguez-Silverio J., Roque-Ramírez B., Borgonio-Cuadra V.M, & Rodríguez-Pérez J.M. (2022). Osteoprotegerin Gene Polymorphisms Are Associated with Subclinical Atherosclerosis in the Mexican Mestizo Population. Diagnostics, 12(6), 1433.

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Chemokine Expression Profiling Using TLDA

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Chemokine transcription was assessed using custom-made TaqMan low-density array (TLDA) microfluidic cards (Applied Biosystems, Foster City, CA) as described previously [20 (link)]. Briefly, 100 μl of reaction mix, containing a 1:1 mixture of cDNA (from ~ 1 μg total RNA) in RNase-free water and 2x TaqMan Universal PCR Master Mix (Thermo Fisher Scientific, Waltham, MA), was loaded onto cards containing the primers and probesets for 32 genes. Cards were centrifuged to distribute the reaction mixture throughout the fluidic system and run on a Prism 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA). Data were analysed using SDS2.2 software and RQ Manager. Relative gene expression was first normalised to the expression of housekeeping gene Tbp, which encodes TATA-binding protein, and then normalised to that of a calibrator selected arbitrarily from the vehicle control group. Fold change of gene expression in LPS-challenged mice compared to vehicle controls was calculated using the ^ΔΔC_T method.

Thomson C.A., McColl A., Graham G.J, & Cavanagh J. (2020). Sustained exposure to systemic endotoxin triggers chemokine induction in the brain followed by a rapid influx of leukocytes. Journal of Neuroinflammation, 17, 94.

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Quantification of MET and HER2 Copy Number

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Genomic DNA was isolated from FFPE tumor specimens and MET and HER2 copy number amplification was performed by a PRISM 7900HT Fast Realtime PCR system (Applied Biosystems). All quantitative PCR reactions were performed in triplicate using the SYBR Green method. The PCR conditions were: preheating at 50 °C for 2 min; 95 °C for 10 min; 40 cycles at 95 °C for 15 s and 60 °C for 1 min. MET and HER2 copy numbers were calculated by comparison to ALB, located at 4q11-q13, and normalized to normal tissue genomic DNA. Primers for MET and HER2 copy number analysis were the following: MET forward, 5′-ATTGGTGATTGCTTGGGTAGTT-3′; MET reverse, 5′-CCTGTGGGTTTACTTTGGTTG-3′; HER2 forward, 5′-GGAGGATGTGCGGCTCG-3′; HER2 reverse, 5′-CATGGTTGGGACTCTTGACCA-3′; ALB forward, 5′-TGAAACATACGTTCCCAAAGAGTTT-3′; ALB reverse, 5′-CTCTCCTTCTCAGAAAGTGTGCATAT-3′. All PCR products were purified using a PCR purification kit and directly sequenced by standard procedures using forward and reverse primers.

Oh D.Y., Jung K., Song J.Y., Kim S., Shin S., Kwon Y.J., Oh E., Park W.Y., Song S.Y, & Choi Y.L. (2017). Precision medicine approaches to lung adenocarcinoma with concomitant MET and HER2 amplification. BMC Cancer, 17, 535.

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Quantitative RT-PCR Analysis of Rcor1 Deficient Cells

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Sorted Mac1⁺Gr1^lo cells from induced Mx1-Cre; Rcor1^flox/− mice and controls treated with poly(I:C) and from primary recipients of Rcor1^−/− and control BM were isolated by FACS. RNA was extracted using TRIzol and treated with DNase. Reverse transcription reactions were performed using Superscript III (Invitrogen, Grand Island, NY, http://www.lifetechnologies.com). Quantitative RT-PCR (qPCR) was performed with an Applied Biosystems PRISM 7900HT Fast Real-Time PCR system using SYBR green PCR master mix (Grand Island, NY, http://www.lifetechnologies.com). Relative abundance of each cDNA was determined according to the standard curve and normalized to 18S RNA levels. The primers used are listed below: 18S_fwd, 5′-CTCAACACGGGAAACCTCAC-3′; 18S_rev, 5′-CGCTCCACCAACTAAGAACG-3′; Gata2_fwd, 5′-CGCCTGTGGCCTCTACTACAA-3′; Gata2_rev, 5′-TTTCTTGCTCTTCTTGGATTTGCT-3′; Hoxa9_fwd, 5′-AACAATGCCGAGAATGAGAGC-3′; Hoxa9_rev, 5′-CGAGTGGAGCGAGCATGTAG-3′; Meis1_fwd, 5′-AAGATACAGGACTTACCATCCTTCA-3′; Meis1_rev, 5′-GTCTATCATGGGCTGCACTATTCT-3′.

Yao H., Goldman D.C., Fan G., Mandel G, & Fleming W.H. (2015). The Corepressor Rcor1 Is Essential for Normal Myeloerythroid Lineage Differentiation. Stem cells (Dayton, Ohio), 33(11), 3304-3314.

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Validation of SVM Classifiers via QRTPCR

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A subset of nine transcripts featured in SVM classifiers were selected for validation with quantitative real time polymerase chain reaction (QRTPCR). First, total RNA was quantitatively converted with High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, San Diego City, CA) to generate single-stranded cDNA (for a 20μL reaction). Aliquots of 20ng of cDNA were analyzed via QRTPCR using the Prism 7900 HT Fast Real-Time PCR system (Applied Biosystems). Statistical analysis was performed using the comparative ΔCT method. All reactions were run in duplicate and normalized against gyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase 1 (HPRT1). For one transcript (GSTM1), QRTPCR analysis was repeated with 75ng cDNA in order to compensate for low signal. The fold change values were compared using independent samples t-tests (p<0.05).

Tylee D.S., Chandler S.D., Nievergelt C.M., Liu X., Pazol J., Woelk C.H., Lohr J.B., Kremen W.S., Baker D.G., Glatt S.J, & Tsuang M.T. (2014). Blood-Based Gene-Expression Biomarkers of Post-Traumatic Stress Disorder among Deployed Marines: A Pilot Study. Psychoneuroendocrinology, 51, 472-494.

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RNA Extraction, Reverse Transcription, and qPCR Quantification

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RNA was extracted using Trizol (Invitrogen) and treated with DNAse (Ambion, Waltham, MA). Reverse transcription reactions were performed using 1 mg-500 ng of total RNA with Superscript III (Invitrogen). qPCR was performed in an Applied Biosystems PRISM 7900HT Fast Real Time PCR system with SYBR green PCR master mix (Applied Biosystems, Waltham, MA) using the same cycling conditions. Relative abundance of each cDNA was determined according to the standard curve and normalized to 18S RNA levels. Samples were diluted 1:50 for analysis of 18S RNA levels and 1:5 for analysis of test genes.

Nechiporuk T., McGann J., Mullendorff K., Hsieh J., Wurst W., Floss T, & Mandel G. (2016). The REST remodeling complex protects genomic integrity during embryonic neurogenesis. eLife, 5, e09584.

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HER2 Copy Number Amplification Assay

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HER2 copy number amplification was performed with a PRISM 7900HT Fast Realtime PCR system (Applied Biosystems). The primer sequences are provided in supplementary Table S1. All reactions were performed in triplicate. The PCR program was as follows: preheating at 50°C for 2 min; 95°C for 10 min; 40 cycles at 95°C for 15 s and 60°C for 1 min; and 1 cycle for melting curve analysis. RNA isolation and cDNA synthesis were performed using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and SuperScriptIII first-strand kit (Invitrogen, Waltham, MA, USA) according to the manufacturers' instructions. The HeLa cell line, which is a HER2-negative cervical cancer cell line, was used as a calibrator for the relative quantification of HER2 expression level. The calculation equation and PCR protocol were the same as those used for copy number analysis.

Oh D.Y., Kim S., Choi Y.L., Cho Y.J., Oh E., Choi J.J., Jung K., Song J.Y., Ahn S.E., Kim B.G., Bae D.S., Park W.Y., Lee J.W, & Song S. (2015). HER2 as a novel therapeutic target for cervical cancer. Oncotarget, 6(34), 36219-36230.

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FFPE Tumor DNA Amplification Analysis

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Genomic DNA was isolated from Formalin-Fixed Paraffin-Embedded (FFPE) tumor specimens, and IRS2 and MYCL copy number amplification was performed by the PRISM 7900HT Fast Real-Time PCR System (Applied Biosystems). All qPCR reactions were performed in triplicate using the SYBR Green method. The PCR conditions were: preheating at 50°C for 2 min; 95°C for 10 min; 40 cycles at 95°C for 15 s, and 60°C for 1 min. IRS2 copy numbers were calculated from a standard curve constructed from normal DNA amplification in comparison to ALB, located at 4q11-q13, and normalized to the normal tissue genomic DNA. Finally, the number of amplification for IRS2 was calculated as follows: copy number of the target gene (IRS2)/copy number of the reference gene (ALB). Primers for IRS2 and MYCL copy number analysis were the following: IRS2 forward, 5′-F CTTTAGTTGGCTGGCTCTGG-3′; IRS2 reverse, 5′-GTTGTCTGCTCCTGCGAATAG-3′; MYCL forward, 5′-GGGTCTGCCTTTT GTTCTTATCT-3′; MYCL reverse, 5′-AAAGGAGGGGACATTAGCAAG-3′; ALB forward, 5′-TGAAACATACGTTCCCAAAGAGTTT-3′; ALB reverse, 5′-CTCTCCTTCTCAGAAA GTGTGCATAT-3′. PCR products were purified using a PCR purification kit and directly sequenced by standard procedures using forward and reverse primers.

Lee M.S., Jung K., Song J.Y., Sung M.J., Ahn S.B., Lee B., Oh D.Y, & Choi Y.L. (2020). IRS2 Amplification as a Predictive Biomarker in Response to Ceritinib in Small Cell Lung Cancer. Molecular Therapy Oncolytics, 16, 188-196.

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Quantitative Analysis of 3T3-L1 miRNA

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Total RNA of 3T3-L1 cells was isolated by using miRVana Isolation Kit according to the manufacturer’s instructions (Ambion). 0.5 μg total RNA from each sample was reverse-transcribed into cDNA using the PrimeScript^™ RT reagent Kit (Takara). The miRNA levels were quantitatively assessed by SYBR Green-based quantitative real-time PCR with gene-specific primers in an Applied Biosystems PRISM 7900HT Fast Real-Time PCR System according to the manufacturer’s instructions (Applied Biosystems). U6 was used as an internal normalization control.

Zhu Y., Zheng G., Wang H., Jia Y., Zhang Y., Tang Y., Li W., Fan Y., Zhang X., Liu Y, & Liu S. (2017). Downregulated miR-29a/b/c during Contact Inhibition Stage Promote 3T3-L1 Adipogenesis by Targeting DNMT3A. PLoS ONE, 12(1), e0170636.

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Chromatin Immunoprecipitation Assay for Astrocytes

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The astrocytes were cross-linked with 1% formaldehyde and then lysed. The chromatin was harvested and fragmented using sonication and then immunoprecipitated with anti-p-PPARγ antibody or anti-acetyl H4 antibody (positive control) or mouse IgG1 (negative control). Purified DNA was amplified using two sets (sets 1 and 2; Table 4) of IR10 or DR1 or glyceraldehyde 3-phosphate dehydrogenase-specific primers and quantified by real-time PCR using SYBR Green PCR master mix and the PRISM 7900 HT Fast Real-Time PCR System (PE Applied Biosystems, Foster City, CA, USA).

Rai A., Tripathi S., Kushwaha R., Singh P., Srivastava P., Sanyal S, & Bandyopadhyay S. (2014). CDK5-induced p-PPARγ(Ser 112) downregulates GFAP via PPREs in developing rat brain: effect of metal mixture and troglitazone in astrocytes. Cell Death & Disease, 5(1), e1033-.

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