Viia7 real time pcr

Manufactured by Thermo Fisher Scientific

Sourced in United States

The ViiA7 real-time PCR system is a laboratory instrument designed for high-performance gene expression and genotyping analysis. It is capable of performing real-time polymerase chain reaction (PCR) experiments with high precision and sensitivity.

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27 protocols using viia7 real time pcr

Real-Time qPCR Amplification Protocol

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Real time QPCR amplification was performed with diluted cDNA (1:4 dilution). Each 10 μl reaction contained 5 μl of SYBR Green reagent, 4 μl of forward and reverse primer mix (0,75 μM each) and 1 μl of 1:4 cDNA. Reactions were performed in technical triplicate with three different biological samples using the ViiA 7 REAL TIME PCR with SYBR Green chemistry (Applied Biosystems). The cycling condition was: 95 °C for 20 s, 40 cycles at 95 °C for 1 s and 60 °C for 20 s, 95 °C for 15 s, 60 °C 1 min, and a gradient from 60 °C to 95 °C for 15 min. All primer pairs were validated by QPCR against a positive (genomic DNA) and negative (water) control.

Lowe E.K., Garm A.L., Ullrich-Lüter E., Cuomo C, & Arnone M.I. (2018). The crowns have eyes: multiple opsins found in the eyes of the crown-of-thorns starfish Acanthaster planci. BMC Evolutionary Biology, 18, 168.

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Quantitative Real-Time PCR Assay

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Total RNA was isolated using the GeneJET RNA purification kit (Thermo Scientific) following manufacturer’s protocol, including an additional 15 minute DNaseI (Roche) treatment to remove DNA contamination. RNA was quantified using a Nanodrop 1000 spectrophotometer (Thermo Scientific). 1 µg of RNA was reverse transcribed into cDNA using random hexamer primers with the QuantiTect Reverse Transcription Kit (Qiagen), according to manufacturer’s protocol. Each qRT-PCR reaction contained 500 nM of each primer pair, 10 ng of cDNA and 1xABsolute qPCR SYBR Green, Rox Mix (Thermo Scientific). Primers were newly designed, extracted from the Real Time PCR primer Data Bank (RTPrimerDB, http://medgen.urgent.be/rtprimerdb/) or obtained from literature^{23 (link), 51 (link), 52 (link)} (Table 1). qRT-PCR reactions were conducted on the ViiA7 Real time PCR (Applied Biosystems) for 15 min at 95 °C, followed by 40 cycles of 15 sec at 95 °C, 30 sec at 60 °C and 30 sec at 72 °C. β-actin was used as housekeeping gene. Data and melting curves were analysed using ViiA7 RUO software and relative expression compared to controls was calculated using the ∆∆Ct method^{53 (link)}.

van der Wijst M.G., van Tilburg A.Y., Ruiters M.H, & Rots M.G. (2017). Experimental mitochondria-targeted DNA methylation identifies GpC methylation, not CpG methylation, as potential regulator of mitochondrial gene expression. Scientific Reports, 7, 177.

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Quantifying FXN-ett RNA Stability

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Total RNA from cell pellets was isolated using the RNeasy Mini Kit (Qiagen). All research involving patient/patient tissues was approved by the IRB at The University of Alabama at Birmingham (IRB Protocols: N160923005 and N160922011). Autopsy specimens of heart were obtained from the FRDA tissue repository maintained at the Veterans Affairs Medical Center in Albany, NY, USA. Frozen heart tissue was homogenized by grinding thoroughly in liquid nitrogen followed by homogenization (Dounce) 50 times in RLT buffer, then the lysate was applied to RNeasy Mini Kit to extract total RNA. All extracted RNA was treated with TURBO DNA-free kit (Thermo Fisher). The qRT-PCR reactions were performed in triplicate with the Power SYBR Green RNA-to-CT 1-Step Kit (Thermo Fisher Scientific). Control reactions without reverse transcriptase (NoRT) were always included. Reactions were performed using StepOne Plus or ViiA 7 Real Time PCR instruments (Applied Biosystems). All primers used for qRT-PCR are listed in Supplementary Material, Table S4.
For mRNA half-life analyses, cells were treated with 5 μg/ml actinomycin D (Sigma-Aldrich). RNA was isolated at t = 0, 0.5, 1, 4 and 8 h and relative expression of FXN-ett RNA was analyzed by qRT-PCR. Myc-mRNA was used as a positive control. Half-life was calculated according to (65 (link)).

Li Y., Li J., Wang J., Zhang S., Giles K., Prakash T.P., Rigo F., Napierala J.S, & Napierala M. (2022). Premature transcription termination at the expanded GAA repeats and aberrant alternative polyadenylation contributes to the Frataxin transcriptional deficit in Friedreich’s ataxia. Human Molecular Genetics, 31(20), 3539-3557.

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Quantifying Gene Expression in FFPE and PBMC Samples

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RNA was extracted from formalin-fixed, paraffin-embedded blocks containing the tissue samples using the RNeasy FFPE Kit (Qiagen, Valencia, CA). RNA samples were converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) for subsequent use in PCR. A similar approach was used to isolate mRNA from PBMCs. Gene expression was measured by performing QRT-PCR on cDNA samples using the TaqMan custom designed array card assay (Applied Biosystems, Foster City, CA) per manufacturer’s protocol (Applied Biosystems; IFNG, Hs00989291_m1; IL1RL1 (ST2), Hs00249384_m1; IL31, Hs01098710_m1; IL33, Hs04931857_m1; IL4, Hs00174122_m1; STAT6, Hs00598625_m1). The assay was performed on the VIIA 7 Real-Time PCR (Applied Biosystems, Foster City, CA). Gene transcript levels were normalized to human GAPDH (Applied Biosystems, Hs99999905_m1) and relative gene expression was calculated using the comparative CT method.

Pelin K., Hilpelä P., Donner K., Sewry C., Akkari P.A., Wilton S.D., Wattanasirichaigoon D., Bang M.L., Centner T., Hanefeld F., Odent S., Fardeau M., Urtizberea J.A., Muntoni F., Dubowitz V., Beggs A.H., Laing N.G., Labeit S., de la Chapelle A, & Wallgren-Pettersson C. (1999). Mutations in the nebulin gene associated with autosomal recessive nemaline myopathy. Proceedings of the National Academy of Sciences of the United States of America, 96(5), 2305-2310.

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Genotyping of Cytokine Gene Variants

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Genotyping of IL-1β-511C/T (rs16944), IL-6-174G/C (rs1800795), TNF-α-1031T/C (rs1799964), and TGFβ1-509T/C (rs1800469) was performed using assay-on-demand TaqMan® SNP genotyping assays according to the manufacturer's instructions (Applied Biosystems, USA). Genotyping was done by the allelic discrimination method (VIC- and FAM-labelled primers). Assay-on-demand TaqMan assays were ordered from Applied Biosystems: Assay ID C___1839943_10 (rs16944), C___1839697_20 (rs1800795), C___7514871_10 (rs1799964), and C___8708473_10 (rs1800469). The reaction was performed in 10 μl volume using a ViiA™7 Real-Time PCR, per manufacturer's instructions (Applied Biosystems, USA). Analyses of amplification products were performed using ViiA™7, ver 1.1. (Applied Biosystems, USA). All the experimental conditions are available on demand.

Alkhuriji A.F., Al Omar S.Y., Babay Z.A., El-khadragy M.F., Mansour L.A., Alharbi W.G, & Khalil M.I. (2020). Association of IL-1β, IL-6, TNF-α, and TGFβ1 Gene Polymorphisms with Recurrent Spontaneous Abortion in Polycystic Ovary Syndrome. Disease Markers, 2020, 6076274.

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QPCR SNP Genotyping for Neurological Traits

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QPCR SNP dual fluorescent labeled probe assays for rs6323, rs1137070, rs5906957, rs6324, rs1799836, rs3027452, rs4633, rs9605030 and rs9606186 were custom designed by Topgen Biotechnology (Topgen Biotech., Kaohsiung, Taiwan). Sequence and labeled dye information of primer and probes as listed in Table S1. Briefly, QPCR SNP performed with 2X AceGT Genotyping Master Mix (Topgen Biotech., Kaohsiung Taiwan) on ViiA™ 7 Real Time PCR (Applied Biosystems, Waltham, MA, USA). QPCR program is 95 °C 5 min, 60 °C 30 s with data collection for pre-reading, 40 cycles of 95 °C 3 s and 60 °C 40 s with data collection each cycle end at 60 °C, final step is 60 °C 30 s with data collection for post-reading. Allelic discrimination plots were analyzed by ViiA™ 7 SW v1.3.

Chen P.H., Wang Y.Y., Lan T.H., Chan L.P, & Yuan S.S. (2021). Genetic and Proteinic Linkage of MAO and COMT with Oral Potentially Malignant Disorders and Cancers of the Oral Cavity and Pharynx. Cancers, 13(13), 3268.

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Relative Quantification of Gene Expression

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Total mRNA was prepared using PAXgene Blood RNA kit (Qiagen). Complementary DNA was synthesized using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Amplification of the gene product was attained on a ViiA™7 Real-Time PCR (Applied Biosystems) using SYBR Green qPCR Mastermix (SA Biosciences, Qiagen) and primer pairs as reported [33 (link), 40 (link)]. Gene expression levels were calculated by the ddCt method and presented as relative quantity to the average expression in the IGF1^hi group.

Erlandsson M.C., Lyngfelt L., Åberg N.D., Wasén C., Espino R.A., Silfverswärd S.T., Nadali M., Jood K., Andersson K.M., Pullerits R, & Bokarewa M.I. (2019). Low serum IGF1 is associated with hypertension and predicts early cardiovascular events in women with rheumatoid arthritis. BMC Medicine, 17, 141.

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Profiling Bacterial DNase Gene Expression

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Assessment of the expression of different DNase genes was carried out using QPCR. Briefly, RNA was isolated from 72 h old PA biofilm culture grown at 25 °C static condition with/without L-Methionine using TRIzol (Life Technologies) as per manufacturer’s protocol. The isolated RNA was reverse transcribed using random hexamers (NEB) and Tetro reverse transcriptase (Bioline) as per standard protocol. The cDNA was diluted and analyzed for the presence different DNase genes using specific primers (Table 1) by QPCR SYBR® FAST qPCR Master Mix (Kapa Biosystems) in an Applied Biosystems® ViiA™ 7 Real time PCR instrument. Expression was normalized to the housekeeping gene 16s rRNA.

Gnanadhas D.P., Elango M., Datey A, & Chakravortty D. (2015). Chronic lung infection by Pseudomonas aeruginosa biofilm is cured by L-Methionine in combination with antibiotic therapy. Scientific Reports, 5, 16043.

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Quantifying Mitochondrial DNA Copy Number

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10 ng of total cellular DNA was used as input for the qPCR. Primers amplifying a nDNA region (β-actin) and a mtDNA region (D-loop) were used (Table 1). For validation, an independent mtDNA primer pair of the mtCOX1 region was used. qPCR reactions were conducted on the ViiA7 Real time PCR (Applied Biosystems) for 15 min at 95 °C, followed by 40 cycles of 15 sec at 95 °C, 30 sec at 60 °C and 30 sec at 72 °C. Data and melting curves were analysed using ViiA7 RUO software. The mtDNA copy number was determined with the formula:

2 ({}^{Ct}n {DNA}^{⁎ primer efficiency} - {}^{Ct}m {tDNA}^{⁎ primer efficiency})

^{54 (link)}. To determine the 7S DNA primer formation, primers amplifying both the mtDNA and 7S DNA (7S DNA A + B1), or only the mtDNA (7S DNA A + B2) were used (Table 1), as described previously^{15 (link)}. The level of 7S DNA was calculated with the formula:

2 ({}^{Ct}7 S DNA A + B 2^{⁎ primer efficiency} - {}^{Ct}7 S DNA A + B 1^{⁎ primer efficiency})

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Quantitative Expression Analysis of HITT

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Total RNA was extracted using Trizol Reagent according to the manufacturer’s instructions (Invitrogen, #15596018). cDNA was synthesized by using PrimeScript reverse transcription (RT) reagent kit (TaKaRa, #RR047A) in presence of gDNA Eriser. Quantitative real-time PCR was carried out in the ViiA7 real-time PCR (Applied Biosystems) using SYBR Premix Ex Taq II kit for Real-Time (Takara, #RR820L). The primer sequences used in RT-PCR are as follows: HITT forward 5′-ACACAAATGCTGGCCTCTGTCA-3′, reverse 5′-GGCAAGTGGCAAAGCCTCTC-3′, 18 s forward 5′-AACTTTCGATGGTAGTCGCCG-3′, reverse 5′-CCTTGGATGTGGTAGCCGTTT-3′, U1 forward 5′-GGGAGATACCATGATCACGAAGGT-3′, reverse 5′-CCACAAATTATGCAGTCGAGTTTCCC-3′.

Zhao K., Wang X., Xue X., Li L, & Hu Y. (2020). A long noncoding RNA sensitizes genotoxic treatment by attenuating ATM activation and homologous recombination repair in cancers. PLoS Biology, 18(3), e3000666.

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