Proflex thermal cycler

Manufactured by Thermo Fisher Scientific

Sourced in United States

The ProFlex Thermal Cycler is a laboratory instrument designed for the amplification of DNA samples through the process of polymerase chain reaction (PCR). It provides precise temperature control and automated cycling capabilities to facilitate the DNA replication process.

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Thermal cycler (242 citations) ProFlex (25 citations) ProFlex PCR System Thermal Cycler (4 citations) ProFlex PCR Thermal Cycler (3 citations) Proflex 2× flat block thermal cycler (2 citations) ProFlex™ Base Thermal Cycler (2 citations)

12 protocols using proflex thermal cycler

Virulence Factors Identification in L. monocytogenes

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From the same DNA extractions used to carry out the identification of the isolates, the presence or absence of nine major virulence factors was determined for L. monocytogenes, as indicated in Table 2 [27 (link)].
The hlyA, actA, inlB and iap genes were detected by single PCRs, while two multiple PCRs were performed for the remaining virulence genes: one for inlA, inlC and inlJ, and another for plcA and prfA. Single PCRs were performed using the same reagent concentrations employed to identify isolates (Table 1). However, the primers used, their concentrations and the thermocycling conditions were those shown in Table 3. In the case of the multiplex PCRs, a final volume of 50 µL was obtained, and the concentrations of MgCl₂ (2 mM), of Taq DNA polymerase (2U), and of each primer used were modified, as can be seen in Table 2.
Amplification reactions were carried out in a ProFlex™ thermal cycler (Applied Biosystems). Results were examined by electrophoresis in 1.5% agarose gel and visualized by means of a UV light transilluminator (Bio-Rad).

Panera-Martínez S., Capita R., García-Fernández C, & Alonso-Calleja C. (2023). Viability and Virulence of Listeria monocytogenes in Poultry. Microorganisms, 11(9), 2232.

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Bone Marrow RNA Profiling

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Total RNA from bone marrow was isolated with Trizol (Invitrogen, San Diego, CA). cDNA was synthesized from l µg of total RNA with superscript III (dN6) (Invitrogen). mRNA levels of IL-7R, PU1, Foxo1, and EBF1 were measured by RT-PCR using a cDNA template and the appropriate primers. RT-PCR was performed with the ProFlex™ Thermal Cycler (Applied Biosystems Foster City, CA) and Ex taq (Takara, Otsu, Japan). Relative levels of PCR products were determined after normalizing to an endogenous GAPDH control.

Lee G.Y., Jeong S.Y., Lee H.R, & Oh I.H. (2019). Age-related differences in the bone marrow stem cell niche generate specialized microenvironments for the distinct regulation of normal hematopoietic and leukemia stem cells. Scientific Reports, 9, 1007.

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ctDNA Detection and Quantification via dPCR

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DNA was extracted utilizing the QIAamp Circulating Nucleic Acid Kit (Qiagen, Manchester, United Kingdom). ctDNA testing via dPCR assay was performed as previously described (2–10 (link)). In brief, targeted-panel sequencing from two FFPE samples was performed using the ABC-Bio or RMH- 200 gene panels and one to two variants were selected for the dPCR assay (11, 12 ). The Thermo Scientific Custom TaqMan SNP Geneotyping Assay design tool was employed for assay design. Assay optimization was performed with a ProFlex Thermal Cycler (Applied BioSystems), Automated Droplet Generator (Bio-Rad, Pleasanton) and Droplet Reader (Bio-Rad, Pleasanton). Two or more FAM-positive droplets were required for the sample to be called positive. If a positive result was obtained, this was independently confirmed on a second sample. Copies/mL and allele fraction were calculated as previously described (13 (link)).

Coakley M., Villacampa G., Sritharan P., Swift C., Dunne K., Kilburn L., Goddard K., Pipinikas C., Rojas P., Emmett W., Hall P., Harper-Wynne C., Hickish T., Macpherson I., Okines A., Wardley A., Wheatley D., Waters S., Palmieri C., Winter M., Cutts R.J., Garcia-Murillas I., Bliss J, & Turner N.C. (2023). Comparison of Circulating Tumor DNA Assays for Molecular Residual Disease Detection in Early-Stage Triple-Negative Breast Cancer. Clinical Cancer Research, 30(4), 895-903.

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Salmonella Identification via 16S rRNA Sequencing

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Salmonella isolates were identified by 16S rRNA sequencing. The reaction mixture in a volume of 20 mL consisted of 10 μL of master mix (DreamTaq Green PCR Master mix 2×, Thermo Fisher Scientific, Waltham, Massachusetts, USA), 1 μL of forward 27F (5’-AGAGTTTGATYMTGGCTCAG-3’), reverse 515R (5’-TTACCGCGGCKGCTGGCAC-3’) universal primer [31 (link)], 1 μL of DNA, and 7 μL of water without nucleases. Amplification was performed at 55°C on a Proflex thermal cycler (Applied Biosystems, USA). Amplification products were visualized by electrophoresis in 1.5% agarose gel (Quantum gel documenting system, Vilber, Collégien, France). The sequencing of bacterial 16S rRNA gene fragments was performed using the Big Dye Terminator v3.1 Cycle Sequencing Kit according to the manufacturer’s protocol (BigDye^® Terminator v3.1 Cycle Sequencing Kit Protocol, Applied Biosystems). The sequencing results were processed using SeqA. The search for homologous nucleotide sequences of the 16S rRNA genes was performed using the BLAST program in the International Gene Bank database of the US National Center for Biotechnology Information (NCBI). Phylogenetic analysis was performed using the MEGA6 software (https://www.megasoftware.net/). Nucleotide sequence alignment was performed using the ClustalW algorithm version 1.6 (http://www.clustal.org/).

Mendybayeva A., Abilova Z., Bulashev A, & Rychshanova R. (2023). Prevalence and resistance to antibacterial agents in Salmonella enterica strains isolated from poultry products in Northern Kazakhstan. Veterinary World, 16(3), 657-667.

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Optimization of 22-STR Panel PCR

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Two different total reaction volumes, 10 and 25 μl, were adjusted to evaluate the performance of this 22-STR panel. The value of each reagent in the 25-μl PCR system was 2.5 times larger than that in the 10-μl system, and the PCR conditions were in accordance with those mentioned above. To evaluate the PCR performance in various PCR thermocyclers, we conducted the PCR in the Applied Biosystems Veriti Thermal Cycler, Applied Biosystems ProFlex Thermal Cycler, Applied Biosystems 9700 Thermal Cycler, and Applied Biosystems 2720 Thermal Cycler (Thermo Fisher Scientific, South San Francisco, CA). The DNA mix was regarded as the DNA template, and the reaction conditions used were as mentioned above.

Cui W., Jin X., Guo Y., Chen C., Zhang W., Wang Y., Lan J, & Zhu B. (2020). Development and Validation of a Novel Five-Dye Short Tandem Repeat Panel for Forensic Identification of 11 Species. Frontiers in Genetics, 11, 1005.

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Standardizing PCR Techniques for Viral Load

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One microliter of cDNA was used in all different PCR approaches in order to equalize the amount of genetic material. The final volume was set to 15μL for all techniques. Conventional PCR was performed using TopTaq Master mix (Qiagen) in a Veriti Thermocycler. Real-time PCR using SYBR Green (Quantifast SYBR Qiagen), EVA Green (Biotium), or probes (Quantitect Virus; Qiagen) were performed in a RealPlex 4 Thermocycler (Eppendorf). Supplies from Applied Biosystems (Foster City, CA, USA) were used to perform digital polymerase chain reaction (dPCR) experiments (Quantstudio 3D 20K chip and Master mix), including equipment (Chip Loader, Proflex Thermal Cycler, and Quantstudio 3D reader). The annealing temperature for all protocols was 61°C, and the extension temperature was 72°C (even for optimized DNA polymerases).
Due to the small input volume required in dPCR, we decided to standardize all techniques using a fixed volume instead of the same concentration of genetic material; the volume used was close to the maximum required for the technique. Additionally, when working with patient samples, particularly large numbers of samples, the use of volume is easier and less laborious than fixing the concentration of genetic material. Sample to sample variation can still exist, however, because viral load fluctuation among samples is frequent.

Esposito D.L.A, & Fonseca B.A.L.D. (2017). Sensitivity and detection of chikungunya viral genetic material using several PCR-based approaches. Revista da Sociedade Brasileira de Medicina Tropical, 50(4).

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Diagnosing Haemotropic Pathogens via PCR

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Several primer sets were used to diagnose haemotropic pathogens (Table 1) and all PCR reactions were carried out in 25 μl reaction volumes, using the Proflex Thermal Cycler (Life Technologies, Carlsbad, California, U.S.A). The positive controls for T. theileri were the samples that tested positive for HCT. On the other hand, for Babesia, T. vivax, and A. margianle, previously prepared plasmids were used (29 (link)–31 (link)).

Chávez-Larrea M.A., Cholota-Iza C., Cueva-Villavicencio J., Yugcha-Díaz M., Ron-Román J.W., Rodríguez-Cabezas A., Saegerman C, & Reyna-Bello A. (2023). Molecular identification of Trypanosoma theileri (Laveran, 1902) in cattle from two slaughterhouses in Ecuador and its relation with other haemotropic agents. Frontiers in Veterinary Science, 10, 1153069.

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Quantifying Mitochondrial DNA in ASCs

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RNA extraction was performed according to manufacturer recommendations (RNeasy Micro Kit, Qiagen) to perform Reverse Transcription (RT) with high-capacity cDNA reverse transcription kit (TerhmoFisher) on ProFlex thermal cycler (Life technologies). Total DNA was extracted from ASC with AllPrep DNA/RNA Mini kit (Qiagen). Mitochondrial DNA was quantified using mitochondrial gene MT-ND1 (NADH-ubiquinone oxidoreductase chain 1). Quantitative polymerase chain reactions (qPCR) was performed with 2 µL of cDNA or DNA, 1.5 µL forward and reverse primers at 2 µM and 5 µL of fast SYBR green master mix (ThermoFisher). PCR cycles were performed in StepOne (ThermoFisher) with PUM or β-actin (genomic DNA gene to normalized mitochondrial DNA quantification) as the housekeeping gene. Analysis was made with the StepOne software, with cycle threshold evaluation (CT). Mean of duplicates CT was used to calculate deltaCT = CT_gene − CT_PUM/b-actine. Data were analyzed in 2^−deltaCT or in fold increase of 2^-deltaCT to control cells. Primer sequences are listed in Supplementary Table 2.

Bourdens M., Jeanson Y., Taurand M., Juin N., Carrière A., Clément F., Casteilla L., Bulteau A.L, & Planat-Bénard V. (2019). Short exposure to cold atmospheric plasma induces senescence in human skin fibroblasts and adipose mesenchymal stromal cells. Scientific Reports, 9, 8671.

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Quantification of Transfected Cell RNA

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Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) from both control and transfected cells (E6/E7, LMP1, and E6/E7 + LMP1) according to the instructions of the manufacturer. For the reverse transcription (RT)-PCR analysis, 100 ng of total RNA was reverse transcribed using SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA). Samples were incubated in the Proflex Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA) for reverse transcription at 60°C for 30 min, initial PCR activation step at 94°C for 2 min followed by 40 PCR cycles. Each cycle consisted of annealing at 94°C for 15 s, at 61°C for 30 s, and at 68°C for 1 min. Final annealing was at 68°C for 5 min. The oligonucleotide primers used in this study have been described previously (45 (link), 51 (link)). The RT-PCR products were examined by electrophoresis on 1% agarose gel containing 0.2 μg/ml ethidium bromide.

Gupta I., Jabeen A., Vranic S., Al Moustafa A.E, & Al-Thawadi H. (2021). Oncoproteins of High-Risk HPV and EBV Cooperate to Enhance Cell Motility and Invasion of Human Breast Cancer Cells via Erk1/Erk2 and β-Catenin Signaling Pathways. Frontiers in Oncology, 11, 630408.

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Profiling miRNA in T2DM and IHD

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The serum of 22 T2DM and 26 T2DM IHD miRNA samples were reverse-transcribed using a miRCURY LNA RT Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions, on a Proflex thermal cycler (Thermo Fisher Scientific, Waltham, USA). Subsequently, the qRT-PCR reaction was performed using specific primers and a miRCURY LNA SYBR Green PCR Kit (Qiagen, Germany). The samples were run on the qPCR plates in duplicate on the LightCycler 480 Real-Time PCR System (Roche, Switzerland). Based on the NormFinder (32 (link)), miR-103a-3p and miR-199b-5p were used as endogenous reference miRNAs. The primers efficiency for targets and reference miRNAs has been checked. After calculating the qPCR efficiency, relative expression levels of miRNAs were calculated using the delta-delta Ct (2^–ΔΔCt) method. Ct is the threshold cycle which is a point when fluorescence reading surpasses a set baseline. This method calculates samples’ relative fold expression, using a reference miRNA as the normalizer (33 (link)).

Bielska A., Niemira M., Bauer W., Sidorkiewicz I., Szałkowska A., Skwarska A., Raczkowska J., Ostrowski D., Gugała K., Dobrzycki S, & Krętowski A. (2022). Serum miRNA Profile in Diabetic Patients With Ischemic Heart Disease as a Promising Non-Invasive Biomarker. Frontiers in Endocrinology, 13, 888948.

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