Tetrad2 thermal cycler

Manufactured by Bio-Rad

Sourced in United States

The Tetrad2 Thermal Cycler is a laboratory instrument used for DNA amplification, also known as PCR (Polymerase Chain Reaction). It provides precise temperature control and cycling for the thermal reactions required in various molecular biology applications.

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Lab products found in correlation

Typhoon fla 9500 phosphorimager, by GE Healthcare (4 mentions) Pwo polymerase, by Roche (4 mentions) Qiaamp dna micro kit, by Qiagen (3 mentions) Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions) Nanodrop 8000 spectrophotometer, by Thermo Fisher Scientific (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions) Qiamp dna micro kit, by Qiagen (1 mentions) Illustra rnaspin mini isolation kit, by GE Healthcare (1 mentions) Superscript first strand kit, by Thermo Fisher Scientific (1 mentions) Primer pairs, by Thermo Fisher Scientific (1 mentions) Quantitect reverse transcription kit, by Qiagen (1 mentions) Dna engine dyad, by Bio-Rad (1 mentions) Qiamp dna blood mini kit, by Qiagen (1 mentions) Applied biosystems steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions) Nucleospin gel and pcr clean up column, by Macherey-Nagel (1 mentions) Jumpstart redtaq readymix, by Merck Group (1 mentions) Mastercycler ep gradient thermal cycler, by Eppendorf (1 mentions) Dneasy plant mini kit, by Qiagen (1 mentions)

11 protocols using tetrad2 thermal cycler

RNA Extraction and cDNA Synthesis Protocol

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Total RNA from the frozen tissues was isolated using TRIzol® reagent (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions, and quantified by measuring the absorbance at 260 nm using a Nanodrop 8000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). The quality of the extracted RNA was determined by measuring the RNA integrity number (RIN) values (Additional file 1 shows a representative figure of RIN measurements) using an Agilent 2100 Bioanalyzer (Santa Clara, CA). Thereafter, first-strand cDNA was synthesized by using the High-Capacity cDNA reverse transcription kit (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. In brief, 1.5 μg of total RNA from each sample was added to a mix of 1.0 μl MultiScribe™ reverse transcriptase (RT), 2.0 μl 10X RT buffer, 0.8 μl 25X dNTP mix (100 mM), 2.0 μl 10X RT random primers, and 4.2 μl nuclease-free water. The final reaction mix was kept at 25 °C for 10 min, heated to 37 °C for 120 min, heated for 85 °C for 5 min, and finally cooled to 4 °C in a Tetrad 2 thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA).

Grant M.K., Seelig D.M., Sharkey L.C, & Zordoky B.N. (2017). Sex-dependent alteration of cardiac cytochrome P450 gene expression by doxorubicin in C57Bl/6 mice. Biology of Sex Differences, 8, 1.

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Telomere Length Measurement by STELA

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DNA was extracted from 3,000 flow-sorted T cells per subset using a QIAmp DNA Micro Kit (Qiagen). STELA was carried out at the XpYp and 17p telomeres as described previously (25 (link)). For each sample, 1 µM of the Telorette-2 linker was added to purified genomic DNA in a final volume of 40 µL. Multiple PCRs were performed for each test DNA in volumes of 10 µL incorporating 1 µL of the DNA/Telorette-2 mix and 0.5 µM of the telomere-adjacent and Teltail primers in 75 mM Tris-HCl pH 8.8, 20 mM (NH₄)₂SO₄, 0.01% Tween-20, and 1.5 mM MgCl₂, with 0.5 U of a 10:1 mixture of Taq (ABGene) and Pwo polymerase (Roche Molecular Biochemicals). The reactions were processed in a Tetrad2 Thermal Cycler (Bio-Rad). DNA fragments were resolved using 0.5% Tris-acetate-EDTA agarose gel electrophoresis and identified via Southern hybridization using a random-primed α-³³P-labeled (PerkinElmer) TTAGGG repeat probe, together with probes specific for the 1 kb (Stratagene) and 2.5 kb molecular weight markers (Bio-Rad). Hybridized fragments were detected using a Typhoon FLA 9500 Phosphorimager (GE Healthcare). The molecular weights of the DNA fragments were calculated using a Phoretix 1D Quantifier (Nonlinear Dynamics).

Roger L., Miners K.L., Leonard L., Grimstead J.W., Price D.A., Baird D.M, & Ladell K. (2023). T cell memory revisited using single telomere length analysis. Frontiers in Immunology, 14, 1100535.

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Quantification of TLR4 mRNA Expression

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Extraction of total RNA from untreated and treated THP1 cells (3 × 10⁶) was performed using the Illustra RNAspin Mini Isolation Kit (GE Healthcare, Little Chalfont, UK). Total RNA was used to monitor the mRNA expression of human TLR4. Complementary DNA (cDNA) was produced from total RNA extracts using the SuperScript first-strand kit (Invitrogen – Life Technologies, Carlsbad, CA, USA) and performed in a Tetrad2 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). Primer pairs (Invitrogen) and the sequences used were: 5′-GAA GCT GGT GGC TGT GGA-3′ (sense) and 5′-TGA TGT AGA ACC CGC AAG-3′ (antisense) for human TLR4, as previously described [33 (link)]. The β-actin primer sequences used were 5′-TGA TGA CAT CAA GAA GGT GGT GAA G-3′ (sense) and 5′-TCC TTG GAG GCC ATG TGG GCC AT-3′ (antisense). Real-time PCR was done using the QuantiTect Reverse Transcription kit containing PCR SyberGreen Master Mix (Qiagen, Hilden, Germany) to quantitatively monitor the mRNA expression of TLR4 compared with β-actin gene expression (a house-keeping gene used as an internal control). For each analysis, a negative control was prepared using all the reagents except the cDNA template. All the reactions were run in triplicate.

El-Obeid A., Alajmi H., Harbi M., Yahya W.B., Al-Eidi H., Alaujan M., Haseeb A., Trivilegio T., Alhallaj A., Alghamdi S., Ajlouni A.W, & Matou-Nasri S. (2020). Distinct anti-proliferative effects of herbal melanin on human acute monocytic leukemia THP-1 cells and embryonic kidney HEK293 cells. BMC Complementary Medicine and Therapies, 20, 154.

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Single Telomere Length Analysis in CD8+ T Cells

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DNA was extracted from 6,000 FACS-purified CD8⁺ T cells per subset using a QIAamp DNA Micro Kit (Qiagen). Single telomere length analysis was carried out at the XpYp telomere as described previously^{67 (link)}. Briefly, genomic DNA was eluted in 35 μl of Tris (10 mM) containing 0.75 μl of the Telorette-2 linker (10 μM). Multiple PCRs were then performed for each test DNA. Each reaction was set up in a final volume of 10 μl containing 1 μl of DNA and 0.5 μM of the telomere-adjacent and teltail primers in 75 mM Tris-HCl pH 8.8, 20 mM (NH₄)₂SO₄, 0.01% Tween-20, and 1.5 mM MgCl₂, with 0.5 U of a 10:1 mixture of Taq (Thermo Fisher Scientific) and Pwo polymerase (Roche Molecular Biochemicals). The reactions were processed in a Tetrad2 Thermal Cycler (Bio-Rad). DNA fragments were resolved via 0.5% Tris-acetate-EDTA agarose gel electrophoresis and identified via Southern hybridization with a random-primed anti-³²P-labeled (PerkinElmer) TTAGGG repeat probe, together with probes specific for molecular weight markers at 1 kb (Stratagene) and 2.5 kb (Bio-Rad). Hybridized fragments were detected using a Typhoon FLA 9500 Phosphorimager (GE Healthcare). The molecular weights of the DNA fragments were calculated using a Phoretix 1D Quantifier (Nonlinear Dynamics).

Galletti G., De Simone G., Mazza E.M., Puccio S., Mezzanotte C., Bi T.M., Davydov A.N., Metsger M., Scamardella E., Alvisi G., De Paoli F., Zanon V., Scarpa A., Camisa B., Colombo F.S., Anselmo A., Peano C., Polletti S., Mavilio D., Gattinoni L., Boi S.K., Youngblood B.A., Jones R.E., Baird D.M., Gostick E., Llewellyn-Lacey S., Ladell K., Price D.A., Chudakov D.M., Newell E.W., Casucci M, & Lugli E. (2020). Two subsets of stem-like CD8+ memory T cell progenitors with distinct fate commitments in humans. Nature immunology, 21(12), 1552-1562.

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Genotyping of Pharmacogenomic Variants

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A 6 mL EDTA blood sample was obtained for each patient and stored at -20°C prior to DNA extraction. Genomic DNA was extracted from the EDTA whole blood using the QIAmp^® DNA blood Minikit (Qiagen^®, Hilden, Germany).
Genotyping of SLCO1B1 and CYP3A5 was carried out using validated and certified TaqMan^®-based real-time PCR methods at Center for Psychopharmacology, Diakonhjemmet Hospital, Oslo, Norway. Designed primers and probes for the detection of SLCO1B1*5 (rs4149056; 521T>C), CYP3A5*2 (rs28365083; 27289C>A), and CYP3A5*3 (rs776746; 6986G>A), were purchased from Applied Biosystems, Foster City, CA. Absence of variant alleles was interpreted as presence of the wild-type allele (*1).
Genotyping of ABCB1 (1199G>A, 1236C>T, 2677G>T, 2677G>A and 3435C>T), POR*28 (rs1057868; C>T), PPARA (rs4253728; G>A) and PPARA (rs4823613; A>G) were performed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) methods, using DNA Engine Dyad^® and Tetrad^® 2 Thermal Cycler (Bio-Rad Laboratories, Inc.). Specific primers and enzymes were used for the different sequence variants. The different PCR products were digested with 1 Unit of the associated restriction enzyme, and digested products were separated by electrophoresis on a 3% agarose gel, visualized under ultraviolet light after staining with GelRed™.

Robertsen I., Åsberg A., Granseth T., Vethe N.T., Akhlaghi F., Ghareeb M., Molden E., Reier-Nilsen M., Holdaas H, & Midtvedt K. (2014). More potent lipid lowering effect by rosuvastatin compared to fluvastatin in everolimus treated renal transplant recipients. Transplantation, 97(12), 1266-1271.

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Quantitative PCR analysis of methane oxidation genes

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Amplification of products by PCR was performed in 25 μL reaction volumes using a BIORAD Tetrad 2 thermal cycler. The reaction mixture was 1× Master Mix (PCR Biosystems, UK), 0.4 μM forward primer and 0.4 μM reverse primer. PCR primers and amplification protocols used to screen for 16S rRNA, mxaF, xoxF1-5 and mdh2 genes are detailed in Additional File 27. PCR products were purified using NucleoSpin Gel and PCR clean-up columns (Macherey-Nagel, Germany) according to the manufacturer’s instructions. The copy number of 16S rRNA, mxaF and xoxF5 genes in DNA and cDNA samples was estimated using quantitative PCR (qPCR) (Applied Biosystems Step one plus real-time PCR system, Thermo Fisher, MA, USA). The reaction mixture was BioLine Sensifast Hi Rox master mix, 0.4 μM each primer and with the addition of bovine serum albumin (0.2 μg). Standards were prepared using xoxF5 and mxaF PCR products amplified from DNA of Methylocella silvestris BL2, diluted to a copy number of 10⁸ to 10¹ per microlitre. Three biological replicates from each environment were tested, each with three technical replicates. The efficiency of the amplification was 98% for mxaF and 83% for xoxF5. A two-way ANOVA test was performed using the R package dplyr to test for significant differences between the test groups.

Macey M.C., Pratscher J., Crombie A.T, & Murrell J.C. (2020). Impact of plants on the diversity and activity of methylotrophs in soil. Microbiome, 8, 31.

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Plastid DNA Sequencing for Plant Identification

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Genomic DNA was extracted from leaf material using the DNeasy Plant MiniKit (Qiagen, Valencia, CA, USA), following the manufacturer’s protocol. We obtained sequences for three DNA plastid regions matK, ndhF, and rbcL, using custom Pooideae-specific primers (Schubert et al., 2019a ; Supplementary Table S2). PCR was performed on a Tetrad 2 Thermal Cycler (Bio-Rad, Hercules, CA, USA) and a Mastercycler ep Gradient Thermal Cycler (Eppendorf, Hamburg, Germany) using JumpStart REDTaq ReadyMix (Sigma-Aldrich, St. Louis, MO, USA) and standard conditions with 58 °C annealing and 2 min extension. PCR products were Sanger-sequenced in both directions using the same primers as for PCR. Chromatograms and sequences were inspected in BioEdit (Hall, 1999 ), and automatic alignments generated with manual adjustments (Supplementary Datasets S1–S3).

Fjellheim S., Young D.A., Paliocha M., Johnsen S.S., Schubert M, & Preston J.C. (2022). Major niche transitions in Pooideae correlate with variation in photoperiodic flowering and evolution of CCT domain genes. Journal of Experimental Botany, 73(12), 4079-4093.

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Thermal Stability-based Protein Profiling

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CETSA were adapted from a previously described protocol (Martinez Molina et al., 2013 (link)). HEK293T cells were lysed in a buffer containing: 50 mM Tris pH = 7.5, 400 mM KCl, 4 mM Mg(OAc)₂, 0.5% Triton X-100 and protease inhibitors (EDTA-free protease inhibitor tablets, Roche). The lysates were clarified at 20,000×g for 15 min at 4°C. The supernatant was then incubated with ISRIB (1 μM, 0.1% DMSO) or DMSO (0.1%) at 30°C for 20 min, and subsequently spun at 100,000×g for 30 min at 4°C to pellet ribosomes. Supernatants following the high-speed spin were divided into PCR tubes and subjected to a gradient of temperatures for 3 min using the thermal cycler's built-in gradient function, such that column one corresponded to 52°C and column 12 corresponded to 62°C (Tetrad 2 Thermal Cycler, Bio-Rad). Samples were allowed to cool for 3 min at room temperature, transferred to microfuge tubes, and spun at 20,000×g for 20 min at 4°C to separate the soluble fraction from the insoluble precipitates. The soluble fraction was then loaded on a 10% SDS-PAGE gel (Bio-Rad) and analyzed by Western blotting as described above.

Sidrauski C., Tsai J.C., Kampmann M., Hearn B.R., Vedantham P., Jaishankar P., Sokabe M., Mendez A.S., Newton B.W., Tang E.L., Verschueren E., Johnson J.R., Krogan N.J., Fraser C.S., Weissman J.S., Renslo A.R, & Walter P. (2015). Pharmacological dimerization and activation of the exchange factor eIF2B antagonizes the integrated stress response. eLife, 4, e07314.

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CDKN2A mRNA Expression Analysis in HGSC

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A subset of 2280 cases had CDKN2A mRNA expression data from NanoString n‐counter analysis. RNA was extracted from 10 μm sections from formalin fixed paraffin embedded (FFPE) tissue blocks, which were macrodissected to avoid adjacent benign tissue but included tumor stroma using the Qiagen miRNeasy (Qiagen Inc. Toronto, Ontario, Canada) FFPE protocol and quantitated on a Nanodrop spectrophotometer (Thermo‐Fisher Scientific, Waltham, MA, USA). After mixing 500 ng of total RNA per sample with a custom codeset (NanoString Technologies Inc, Seattle, WA, USA) and hybridization buffer (NanoString), hybridization was performed using a Tetrad 2 thermal cycler (Bio Rad Laboratories Inc, Hercules, CA, USA) for 16 or 20 h and then analyzed on a nCounter Digital Analyzer (NanoString). Data was normalized to housekeeping genes (RPL19, ACTB, PGK1, SDHA, and POLR1B) and pre‐processed to a reference of 3 pooled ovarian cancer specimens as described previously 21. We interrogated the cBioportal for associations of CDKN2A alterations with OS in HGSC from TCGA 22, 23.

Rambau P.F., Vierkant R.A., Intermaggio M.P., Kelemen L.E., Goodman M.T., Herpel E., Pharoah P.D., Kommoss S., Jimenez‐Linan M., Karlan B.Y., Gentry‐Maharaj A., Menon U., Polo S.H., Candido dos Reis F.J., Doherty J.A., Gayther S.A., Sharma R., Larson M.C., Harnett P.R., Hatfield E., de Andrade J.M., Nelson G.S., Steed H., Schildkraut J.M., Carney M.E., Høgdall E., Whittemore A.S., Widschwendter M., Kennedy C.J., Wang F., Wang Q., Wang C., Armasu S.M., Daley F., Coulson P., Jones M.E., Anglesio M.S., Chow C., de Fazio A., García‐Closas M., Brucker S.Y., Cybulski C., Harris H.R., Hartkopf A.D., Huzarski T., Jensen A., Lubiński J., Oszurek O., Benitez J., Mina F., Staebler A., Taran F.A., Pasternak J., Talhouk A., Rossing M.A., Hendley J., Edwards R.P., Fereday S., Modugno F., Ness R.B., Sieh W., El‐Bahrawy M.A., Winham S.J., Lester J., Kjaer S.K., Gronwald J., Sinn P., Fasching P.A., Chang‐Claude J., Moysich K.B., Bowtell D.D., Hernandez B.Y., Luk H., Behrens S., Shah M., Jung A., Ghatage P., Alsop J., Alsop K., García‐Donas J., Thompson P.J., Swerdlow A.J., Karpinskyj C., Cazorla‐Jiménez A., García M.J., Deen S., Wilkens L.R., Palacios J., Berchuck A., Koziak J.M., Brenton J.D., Cook L.S., Goode E.L., Huntsman D.G., Ramus S.J, & Köbel M. (2018). Association of p16 expression with prognosis varies across ovarian carcinoma histotypes: an Ovarian Tumor Tissue Analysis consortium study. The Journal of Pathology: Clinical Research, 4(4), 250-261.

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Telomere Length Analysis of Human SGSCs

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DNA was extracted from human SGSCs using a QIAamp DNA Micro Kit (Qiagen). Single telomere length analysis (STELA) was carried out at the XpYp telomere as described previously by Capper et al 22. Briefly, 1 μM Telorette 2 linker was added to 10 ng of purified genomic DNA in a final volume of 40 μl per sample. Multiple polymerase chain reactions (PCRs) were performed for each test DNA in 10‐μl volumes, incorporating 250 pg of DNA, 0.5 μM telomere‐adjacent and Teltail primers, 75 mM Tris HCl (pH 8.8), 20 mM (NH₄)₂SO₄, 0.01% Tween 20, 1.5 mM MgCl₂, and 0.5 units of a 10:1 mixture of Taq (ABgene) and Pwo polymerase (Roche Molecular Biochemicals). The reactions were processed in a Tetrad2 Thermal Cycler (Bio‐Rad). DNA fragments were resolved by 0.5% Tris–acetate–EDTA agarose gel electrophoresis and identified by Southern hybridization with a random‐primed a‐³³P‐labeled (PerkinElmer) TTAGGG repeat probe, together with probes specific for the 1 kb (Stratagene) and 2.5 kb (Bio‐Rad) molecular weight markers. Hybridized fragments were detected using a Typhoon FLA 9500 Phosphorimager (GE Healthcare). The molecular weights of the DNA fragments were calculated using a Phoretix 1D Quantifier (Nonlinear Dynamics).

Pringle S., Wang X., Verstappen G.M., Terpstra J.H., Zhang C.K., He A., Patel V., Jones R.E., Baird D.M., Spijkervet F.K., Vissink A., Bootsma H., Coppes R.P, & Kroese F.G. (2018). Salivary Gland Stem Cells Age Prematurely in Primary Sjögren's Syndrome. Arthritis & Rheumatology (Hoboken, N.j.), 71(1), 133-142.

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