Prism 7900ht real time pcr system

Manufactured by Thermo Fisher Scientific

Sourced in United States, Japan

The Prism 7900HT Real-Time PCR System is a high-throughput, real-time PCR instrument designed for nucleic acid amplification and detection. It features a 384-well sample capacity and supports fluorescence-based detection methods.

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7900HT Real-Time PCR System (574 citations) 7900HT system (158 citations) ABI Prism 7900HT Fast Real-Time PCR System (130 citations) Prism 7900HT (73 citations) ABI Prism 7900HT Real-Time PCR System (60 citations) 7900HT PCR system (30 citations) PRISM 7900HT system (23 citations) Real-Time System (23 citations) PRISM 7900HT Fast Real-Time PCR System (22 citations) ABI Prism 7900HT Fast Real-Time PCR sequence detection system (9 citations)

13 protocols using prism 7900ht real time pcr system

Germline DNA Extraction and EGFR Mutation Detection

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Germline DNA was extracted from either whole blood or normal lung tissue, while tumor DNA was extracted from the matched tumor tissue. Genotyping of the rs2736100 was conducted using a Taqman-based assay (Life Technologies, CA, USA) in a PRISM 7900HT real-time PCR system (Life Technologies, CA, USA) according to the manufacturer's instruction. EGFR mutations (exons 18 to 21) were detected using Sanger sequencing with a protocol previously established in the lab (29 (link)). EGFRmut+ NSCLC patients were defined as individuals with any somatic mutation detected in exons 18–21 of the tumor DNA, while EGFRmut− patients were individuals with wild-type EGFR in their tumor.

Wei R., Cao L., Pu H., Wang H., Zheng Y., Niu X., Weng X., Zhang H., Favus M., Zhang L., Jia W., Zeng Y., Amos C.I., Lu S., Wang H.Y., Liu Y, & Liu W. (2015). TERT Polymorphism rs2736100-C Is Associated with EGFR Mutation-Positive Non-Small Cell Lung Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 21(22), 5173-5180.

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Quantification of Telomere Length in NSCLC

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Blood DNA of NSCLC patients (n=351) and part of healthy controls (n=343) were used to quantify the mean LTL using our previously established real-time PCR based protocol (27 (link)). Briefly, mean LTL telomere length was quantified as the quantity of telomere repeats relative to that of the RNase P gene as a reference, using the primers and conditions described before (27 (link)). Reactions were performed in duplicates in 10 μl reactions in the same plate with a PRISM 7900HT real-time PCR system (Life Technologies). The mean LTL was defined as a T/S ratio between telomere repeats and the RNase P gene for each sample. The T/S ratios were further log transformed (+log10) for subsequent analyses.

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Quantitative Detection of Viral Transcripts

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Total RNA was purified from the spleen, BM, and thymus of FV-infected mice using RNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) and cDNA synthesis was performed by using PrimeScript RT reagent Kit (Takara Bio Inc., Shiga, Japan). The viral DNA fragments were amplified from 0.5 µg or 1 µg of total cDNA and were quantified using Platinum Quantitative PCR SuperMix-UDG with ROX (Life Technologies, Carlsbad, CA) and a Prism 7900HT Real-Time PCR system (Life Technologies). PCR primers and TaqMan probes for the differential detection of F-MuLV and SFFV cDNAs were designed on the env portion of each provirus: primers 5′-AAGTCTCCCCCCGCCTCTA-3′ and 5′-AGTGCCTGGTAAGCTCCCTGT-3′, and a FAM-labeled probe 5′-ACTCCCACATTGATTTCCCCGTCC-3′ for the detection of F-MuLV, and primers 5′-TCTAACCTCACCAACCCTGAT-3′ and 5′-TTTTAGGGCAATGGTATGATTAAAATAA-3′, and a FAM-labeled probe 5′-CCTAGTGTCTGGACCCCCCTATTACGAGG-3′ for the detection of SFFV [59] (link). After initial incubations at 50°C for 2 min and 95°C for 10 min, 40 cycles of amplification were carried out at 95°C for 30 sec and at 58°C for 1 min. A TaqMan rodent GAPDH control reagent (Life Technologies) was used as an internal control. Standard curves obtained by using plasmids containing the env gene of each virus as templates were linear over a range of 10–10⁶ copies in the above reaction.

Takamura S., Kajiwara E., Tsuji-Kawahara S., Masumoto T., Fujisawa M., Kato M., Chikaishi T., Kawasaki Y., Kinoshita S., Itoi M., Sakaguchi N, & Miyazawa M. (2014). Infection of Adult Thymus with Murine Retrovirus Induces Virus-Specific Central Tolerance That Prevents Functional Memory CD8+ T Cell Differentiation. PLoS Pathogens, 10(3), e1003937.

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Transcriptional Profiling of FCGRT in Schizophrenia

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RNA was extracted and cDNA synthesized from DLPFC tissue of people with schizophrenia (n=37) and healthy controls (n=37) as previously described in Weickert et al.^{39 (link)} Transcript levels were measured by qPCR using Applied Biosystems’ Prism 7900HT Real-time PCR system (Foster City, CA, USA). A pre-designed Taqman gene expression assay from Applied Biosystems (Foster City, CA, USA) was used for FCGRT (Hs01108967_m1), normalized to the geometric mean of four housekeeper genes; β-actin (Hs99999903_m1), GAPDH (Hs99999905_m1), TATA box binding protein (Hs00427620_m1), and ubiquitin C (Hs00824723_m1) that did not vary in expression between diagnostic groups.^{39 (link)}

Glass L.J., Sinclair D., Boerrigter D., Naude K., Fung S.J., Brown D., Catts V.S., Tooney P., O'Donnell M., Lenroot R., Galletly C., Liu D., Weickert T.W, & Shannon Weickert C. (2017). Brain antibodies in the cortex and blood of people with schizophrenia and controls. Translational Psychiatry, 7(8), e1192-.

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Quantifying HIPK2 mRNA Expression

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Total RNA from the various cell lines was isolated using the RNeasy extraction method (Qiagen, Valencia, CA, USA). First-strand cDNA was synthesized from total RNA by iScript cDNA synthesis (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Taqman RT–PCR analysis was performed on cDNA in a 384-well plate, using a Prism 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Primers and Taqman probes for human HIPK2 and human GUSB were purchased from Applied Biosystems. The expression of target gene in each sample was assayed in triplicate and normalized to human GUSB for mRNA expression analysis. Decreased transcriptional levels of HIPK2 were calculated by dividing the transcriptional levels measured in HIPK2 siRNA samples from those in the control samples.

Dai Y., Kyoyama H., Yang Y.L., Wang Y., Liu S., Wang Y., Mao J.H., Xu Z., Uematsu K., Jablons D.M, & You L. (2021). A novel isoform of Homeodomain-interacting protein kinase-2 promotes YAP/TEAD transcriptional activity in NSCLC cells. Oncotarget, 12(3), 173-184.

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Quantitative Analysis of miRNA Expression

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The cells were inoculated into 96-well plates, and Trizol was added into each well to extract the total RNA. MMLV was used to reverse-transcribe cDNA and the concentration of DNA was detected. The primers for miR-939 and miR-376a were constructed using cDNA as template, and a TaqMan probe set for Muc5ac (Mm01276718-m1; Applied Biosystems, Foster City, CA, USA), and real-time PCR Master Mix (Toyobo Biotech, Osaka, Japan) with a Prism 7900HT Real-Time PCR System (Applied Biosystems) were used to detect the target genes. GAPDH was used as an internal reference. The mRNA expression of miR-939 and miR-376a was detected by RT-PCR. The PCR cycle conditions were: 94°C for 60 s, 37°C for 60 s, and 72°C for 2 min, and 28 cycles were used.

Lin Y., Zhou Z., Xie L., Huang Y., Qiu Z., Ye L, & Cui C. (2022). Effects of miR-939 and miR-376A on ulcerative colitis using a decoy strategy to inhibit NF-κB and NFAT expression. European Journal of Histochemistry : EJH, 66(1), 3316.

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Quantitative RT-PCR Analysis of MCL1 and CCL2

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Total RNA from the various cell lines was isolated using the RNeasy extraction method (Qiagen, Valencia, CA, USA). First-strand cDNA was synthesized from total RNA by iScript cDNA synthesis (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Taqman RT–PCR analysis was performed on cDNA in a 384-well plate, using a Prism 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Primers and Taqman probes were purchased from Applied Biosystems. The expression of target genes MCL1 (Hs03043899_m1, Thermo fisher) and CCL2 (Hs00234140_m1, Thermo fisher) in each sample was assayed in triplicate and normalized to human GUSB for mRNA expression analysis. An unpaired t test was used to calculate statistical significance.

Lee J.C., Liu S., Wang Y., Liang Y, & Jablons D.M. (2022). MK256 is a novel CDK8 inhibitor with potent antitumor activity in AML through downregulation of the STAT pathway. Oncotarget, 13, 1217-1236.

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Quantifying Airway Mucin Expression

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Total RNA was extracted from the lung after OVA challenge with an RNeasy Mini Kit (n = 5 per group; QIAGEN, MD). cDNA was generated using a SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific). Quantitative real-time RT-PCR was performed using a TaqMan probe set for Muc5ac (Mm01276718-m1; Applied Biosystems, CA) and Realtime PCR Master Mix (Toyobo, Japan) with a Prism 7900HT Real-Time PCR System (Applied Biosystems, CA). Expression of Muc5ac was determined relative to the expression of GAPDH.

Miyake T., Miyake T., Sakaguchi M., Nankai H., Nakazawa T, & Morishita R. (2017). Prevention of Asthma Exacerbation in a Mouse Model by Simultaneous Inhibition of NF-κB and STAT6 Activation Using a Chimeric Decoy Strategy. Molecular Therapy. Nucleic Acids, 10, 159-169.

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Quantification of Cul4A and Gli1 mRNA

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Total RNA from the various cell lines was isolated using the RNeasy extraction method (Qiagen, Valencia, CA, USA). First-strand cDNA was synthesized from total RNA by iScript cDNA synthesis (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Taqman RT–PCR analysis was performed on cDNA in a 384-well plate, using a Prism 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Primers and Taqman probes for human Cul4A, human Gusb and human Gli1 were purchased from Applied Biosystems. The expression of target gene in each sample was assayed in triplicate and normalized to human GUSB for mRNA expression analysis. Decreased transcriptional levels of Cul4A or Gli1 were calculated by dividing the transcriptional levels measured in Cul4A siRNA samples from those in the control samples.

Yang Y.L., Ni J., Hsu P.C., Mao J.H., Hsieh D., Xu A., Chan G., Au A., Xu Z., Jablons D.M, & You L. (2015). Cul4A overexpression associated with Gli1 expression in malignant pleural mesothelioma. Journal of Cellular and Molecular Medicine, 19(10), 2385-2396.

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Compound 8a Modulates HIF1α Pathway

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N2a mouse neuroblastoma cells were treated with either vehicle (DMSO + PBS), Compound 8a (at two different concentration 2 μM and 5 μM, prepared in DMSO), 100 μM L-Asc (prepared in 50 mM PBS), 100 μM D-Asc (prepared in 50 mM PBS), and the combinations of 5 μM Compound 8a with 100 μM L-Asc and D-Asc for 4 h. The lysates were used to isolate total mRNA using TRI reagent according to the manufacturer’s protocol (Sigma-Aldrich). Reverse transcription of total RNA was performed using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). The cDNA was diluted, and about 20 ng was used to amplify in an ABI prism 7900 HT Real-time PCR system (Applied Biosystems) for HIF1α, its target genes VEGF, HO-1, and Nrf2 target gene NQO-1 (as a negative control) using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific). Cycling parameters were 95 °C for 10 s, followed by 60 °C for 1 min. Relative expression was calculated using the ΔΔCt method [26 (link)]. The experiment was performed in triplicate. One way ANOVA with post-hoc Tukey test was used for the determining the statistical significance among different groups. Values are expressed as a fold change from control reaction and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin expression. Mean ± SEM is plotted as bar graph.

Osipyants A.I., Poloznikov A.A., Smirnova N.A., Hushpulian D.M., Khristichenko A.Y., Chubar T.A., Zakhariants A.A., Ahuja M., Gaisina I.N., Thomas B., Brown A.M., Gazaryan I.G, & Tishkov V.I. (2017). L-ascorbic acid: A true substrate for HIF prolyl hydroxylase?. Biochimie, 147, 46-54.

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