Viia 7 system

Manufactured by Thermo Fisher Scientific

Sourced in United States, China, Switzerland, United Kingdom, Germany, Australia, Canada, Japan

The ViiA 7 system is a real-time PCR instrument designed for quantitative gene expression analysis and genetic variation detection. It features a 384-well block format and supports a wide range of fluorescent dyes and chemistries.

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ViiA 7TM Real-time PCR System

372 protocols using viia 7 system

Quantifying T-cell receptor rearrangements

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Genomic DNA was extracted from DN3 or DP cells using the DNeasy Blood and Tissue kit (Qiagen). To measure Tcrb rearrangements in DN3 cells, a Taqman PCR assay was used to quantify Vβ-to-DJβ1.1 and Vβ-to-DJβ2.1 rearrangement levels with a primer specific for each Vβ paired and a probe, FAM or HEX, specific for Jβ1.1 or Jβ2.1, respectively. Taqman PCR was performed with conditions according to the manufacturer’s instructions (IDT DNA) on the ViiA 7 system (Applied Biosystems). PCR of CD19 was used for normalization. Primers, probes, and reaction conditions are as described (20 (link)). To assay Tcrb repertoire, DNA from DN3 thymocytes were sent to Adaptive Biotechnologies, who used multiplex PCR to amplify and deep sequence Vβ-Dβ-Jβ rearrangements. Gene segment usage was analyzed by ImmunoSEQ Analyzer software (Adaptive Biotechnologies). To assess Tcra rearrangements in DP cells, representative Vα-Jα rearrangements were quantified using a QuantiFast SYBR Green PCR kit (Qiagen) on the ViiA 7 system (Applied Biosystems) as described (21 (link), 22 (link)). PCR of β2M was used for normalization. We quantified Vα14-Jα18 rearrangments as described (23 (link)).

Burn T.N., Miot C., Gordon S.M., Culberson E.J., Diamond T., Kreiger P.A., Hayer K.E., Bhattacharyya A., Jones J.M., Bassing C.H, & Behrens E.M. (2022). The RAG1 Ubiquitin Ligase Domain Stimulates Recombination of TCR β and α Genes and Influences Development of αβ T Cell Lineages. Journal of immunology (Baltimore, Md. : 1950).

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Quantifying Gene Expression by RT-PCR

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RNA was isolated from the (co)cultured cell lysates with the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), treated with 0.1 U/μl DNAse (Invitrogen) and reverse transcribed into cDNA using 10 U/μl Superscript II (Invitrogen) and random hexamer primers. The PCR was performed using TaqMan Universal Mastermix (Thermofisher Waltham, Massachusetts, USA). Primers and probes were designed and chosen using ProbeFinder Software and the Universal Probe library (Roche Applied Science, Indianapolis, IN, USA) and are listed in Additional file 3. The qPCR reaction per gene included 10 ng μl of cDNA, 1x l TaqMan Universal Mastermix, 10 pmol forward primer, 10 pmol reverse primer, and 0.012 μM probe. The RT-PCR was performed with the Viia7 System (Applied Biosystems, Waltham, MA, USA), with an initial cycle at 50 °C for 2 min and a heating cycle at 95 °C for 10 min, followed by 45 cycles of 30 s at 95 °C and 30 s at 60 °C. Hypoxanthine phosphoribosyltransferase (HPRT) was used to normalize transcript levels. RT-PCR was performed with the Viia7 System and analyzed using QuantStudio Real-Time PCR Software version 1.3 (Applied Biosystems).

Dankers W., den Braanker H., Paulissen S.M., van Hamburg J.P., Davelaar N., Colin E.M, & Lubberts E. (2021). The heterogeneous human memory CCR6+ T helper-17 populations differ in T-bet and cytokine expression but all activate synovial fibroblasts in an IFNγ-independent manner. Arthritis Research & Therapy, 23, 157.

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Quantitative Analysis of RNA Expression

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Total RNA was prepared from cells by using an RNeasy mini kit (Qiagen). For quantitation of HCV-RNA, quantitative RT-PCR was performed by using TaqMan EZ RT-PCR Core Reagents and a ViiA7 system (Life Technologies) according to the manufacturer’s protocol. For quantitation of gene expression, the synthesis of the first-stranded cDNA was performed by using a PrimeScript RT Reagent Kit (Perfect Real Time) (Takara Bio) and quantitative RT-PCR was performed by using Platinum SYBR Green qRT-PCR SuperMix UDG (Life Technologies) according to the manufacturer’s protocol. ApoB, ApoE and MTTP were amplified using the primer pairs described previously [28 (link)]. For quantitation of miRNA, total RNA was prepared from cells by using an miRNeasy mini kit (Qiagen) and miR-122 was determined by using miR-122-specific RT primers and amplified by using specific primers provided in the Taqman MicroRNA Assays (Life Technologies) according to the manufacturer’s protocol. U6 small nuclear RNA (snRNA) was used as an internal control. Fluorescent signals were analyzed by using a ViiA7 system (Life Technologies).

Ono C., Fukuhara T., Motooka D., Nakamura S., Okuzaki D., Yamamoto S., Tamura T., Mori H., Sato A., Uemura K., Fauzyah Y., Kurihara T., Suda T., Nishio A., Hmwe S.S., Okamoto T., Tatsumi T., Takehara T., Chayama K., Wakita T., Koike K, & Matsuura Y. (2017). Characterization of miR-122-independent propagation of HCV. PLoS Pathogens, 13(5), e1006374.

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Quantification of miR-125a/b and FLT3-ITD in AML

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Peripheral blood samples were collected from 14 AML patients with informed consents at Queen Mary Hospital, Hong Kong. Total peripheral blood mononuclear cells were purified according to a previous protocol.²¹ The samples were confirmed to have at least 38% of leukaemia blasts. Total RNA extraction was carried out using TRIzol (ThermoFisher) based on the manufacturer's instructions. The quality and quantity of RNA samples were evaluated by NanoDrop analysis (ThermoFisher) and agarose gel electrophoresis. RNA samples were reverse transcribed into cDNAs using a reverse transcription kit (ThermoFisher) according to the manufacturer's protocol. Taqman® miRNA assays (ThermoFisher) were used to quantify the levels of miR‐125a and miR‐125b, and U6b RNA was used as the internal control. Ssofast Green qPCR kit (Bio‐Rad) was used to quantify the levels of FLT3‐ITD mRNAs, relative to the level of GAPDH. VIIA(TM) 7 System (Applied Biosystems, USA) was used to process all qPCR reactions.

Chen H., Jayasinghe M.K., Yeo E.Y., Wu Z., Pirisinu M., Usman W.M., Pham T.T., Lim K.W., Tran N.V., Leung A.Y., Du X., Zhang Q., Phan A.T, & Le M.T. (2022). CD33‐targeting extracellular vesicles deliver antisense oligonucleotides against FLT3‐ITD and miR‐125b for specific treatment of acute myeloid leukaemia. Cell Proliferation, 55(9), e13255.

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Quantifying Amoebic Gill Disease Pathogen

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Gill swabs were stored in the −80 °C freezer until DNA extraction followed by qPCR analysis to measure the load of N. perurans on the gills. For DNA extraction, swabs were shaken using a FastPrep-24TM 5G lysis system (MP Biomedicals) at 6 m/s for 40 sec. The samples were then transferred to a clean tube and spun down for 10 min at 14,000 rpm to form a pellet. DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen, VIC, Australia) as per manufacturer’s protocol. Samples were normalised to 30 ng/µL. The qPCR assays were run in a ViiATM 7 system (Applied Biosystems, Thermo Fisher Scientific, VIC, Australia) in 384 well plates.
The qPCR assay targeted the 18S rRNA gene of N. perurans (PERU) and the endogenous control gene (ELF) using previously described methods [20 (link),21 (link)]. A plasmid standard curve of N. perurans and NTC (no template control) was run on each qPCR plate. Assays were done in triplicate for PERU and in duplicate for ELF and NTC. Quantity of N. perurans was calculated based on 30 ng of template DNA.

Hudson J., Adams M., Jantawongsri K., Dempster T, & Nowak B.F. (2022). Evaluation of Low Temperature and Salinity as a Treatment of Atlantic Salmon against Amoebic Gill Disease. Microorganisms, 10(2), 202.

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Real-time PCR Gene Expression Analysis

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Real-time PCR was carried out by using a VIIA(TM) 7 System (Applied Biosystems, Foster City, CA, USA). The gene expressions were calculated by normalization against GAPDH. The sequences of primers used are listed in Table S1 and Table S2).

Sun H.J., Cao L., Zhu M.Y., Wu Z.Y., Shen C.Y., Nie X.W, & Bian J.S. (2020). DR-region of Na+/K+-ATPase is a target to ameliorate hepatic insulin resistance in obese diabetic mice. Theranostics, 10(14), 6149-6166.

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Quantification of Liver Gene Expression

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Total RNA was isolated from human hepatocytes and snap‐frozen mouse liver with an RNeasy Mini Kit (Qiagen). cDNA was synthesized with a high‐capacity cDNA Reverse Transcription Kit (Applied Biosystems) and random primers. The mRNA expression levels of genes of interest were analyzed via TaqMan real‐time PCR in a ViiATM7 System (Applied Biosystems). The TaqMan Gene Expression assays used were Mm01263610_m1 (for mouse Pcsk9), Mm00662319_m1 (mouse Fasn), Mm00443090_m1 (mouse Pklr), Mm00504420_m1 (mouse Pnpla3), Mm01282499_m1 (mouse Hmgcr), Hs00545399_m1 (human PCSK9), Hs01005622_m1 (human FASN), Hs00176075_m1 (human PKLR), Hs00228747_m1 (human PNPLA3), and Hs00168352_m1 (human HMGCR) (all from Applied Biosystems). Hprt (mouse Mm03024075_m1) and GADPH (human Hs02758991_g1) (Applied Biosystems) were used as internal controls.

Lee S., Zhang C., Liu Z., Klevstig M., Mukhopadhyay B., Bergentall M., Cinar R., Ståhlman M., Sikanic N., Park J.K., Deshmukh S., Harzandi A.M., Kuijpers T., Grøtli M., Elsässer S.J., Piening B.D., Snyder M., Smith U., Nielsen J., Bäckhed F., Kunos G., Uhlen M., Boren J, & Mardinoglu A. (2017). Network analyses identify liver‐specific targets for treating liver diseases. Molecular Systems Biology, 13(8), 938.

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Intestinal Gene Expression Analysis

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The mRNA expressions of inflammatory factors, 4 anti-microbial peptides, α-defensin 5, lysozyme, angiogenin 4 (ANG 4) and regenerating islet derived 3-gamma (Reg IIIγ), and ER stress markers in the intestine were measured by real-time PCR. Briefly, total RNA was extracted from the small intestine mucosa with Trizol (Invitrogen, Carlsbad, CA, United States); the concentrations of total RNA were determined spectrophotometrically (NanoDrop-2000, Thermo Fisher Scientific, Waltham, MA, United States), and cDNA was synthesized with the ExScript^TM RT-PCR kit (TaKaRa, Dalian, China). Real-time PCR was performed on the ViiA^TM 7 System (Applied Biosystems, Foster City, CA, United States) utilizing the following thermal cycling conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. Primer sequences were chosen according to our previous studies (Liu et al., 2016 (link); Guo et al., 2017 (link)) and listed in Supplementary Data (Table S1).

Guo X., Tang R., Yang S., Lu Y., Luo J, & Liu Z. (2018). Rutin and Its Combination With Inulin Attenuate Gut Dysbiosis, the Inflammatory Status and Endoplasmic Reticulum Stress in Paneth Cells of Obese Mice Induced by High-Fat Diet. Frontiers in Microbiology, 9, 2651.

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Total RNA Extraction and qPCR Analysis

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Total RNA extraction was performed with TRIzol (Life Technologies, USA) according to the manufacturer's instructions, and then RevertAid First-Strand cDNA Synthesis Kit (Thermo Scientific, USA) was used for reverse transcription. Following, GoTaq® quantitative PCR (qPCR) Master Mix (Promega, USA) was used for quantitative PCR with indicated primers on a VIIA(TM) 7 System (Applied Biosystems). Data were analyzed by normalization against GAPDH. The primers used are indicated as in Table 1.

Xiong S., Sun H.J., Cao L., Zhu M., Liu T., Wu Z, & Bian J.S. (2019). Stimulation of Na+/K+-ATPase with an Antibody against Its 4th Extracellular Region Attenuates Angiotensin II-Induced H9c2 Cardiomyocyte Hypertrophy via an AMPK/SIRT3/PPARγ Signaling Pathway. Oxidative Medicine and Cellular Longevity, 2019, 4616034.

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Comprehensive RNA and Protein Analysis of Mouse Tissues

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Total RNA from mouse tissues was extracted with miRNeasy Mini Kit (Qiagen). RT-PCR was performed using M-MLV Reverse Transcriptase kit (Promega) with random primers. Real-time PCR was performed on ViiA^Tm7 system (Applied Biosystems) using SYBR Green PCR Master Mix (Bioline). Gene expression was quantified by the comparative threshold cycle (ΔΔCT) method and results were normalized to the expression of Rpl23 RNA. The primers for Real-time PCR are listed in Supplementary Data 1. Western blotting was performed to detect target proteins using HuR (1:1000, sc-5261, Santa Cruz Biotechnology), GAPDH (1:5000, ab8245, Abcam), UCP1 (1:2000, ab10983, Abcam), PGC1α (1:1000, sc-13067, Santa Cruz Biotechnology), DESMIN (1:1000, MA1-06401, Thermo Fisher Scientific), CIDEA (1:1000, sc-366814, Santa Cruz Biotechnology), phosphorylated (p)-AKT (1:1000, 4058S, Cell Signaling), AKT (1:2000, 9272S, Cell Signaling), INSIG1 (1:1000, sc-98984, Santa Cruz), C/EBPbeta (1:2000, ab32358, Abcam), HMGB1 (1:1000, ab79823, Abcam), and Beta-Actin (1:5000, A1978, Sigma-Aldrich) antibodies.

Siang D.T., Lim Y.C., Kyaw A.M., Win K.N., Chia S.Y., Degirmenci U., Hu X., Tan B.C., Walet A.C., Sun L, & Xu D. (2020). The RNA-binding protein HuR is a negative regulator in adipogenesis. Nature Communications, 11, 213.

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