Simpleprobe probes

Manufactured by TIB Molbiol

Sourced in Germany

SimpleProbe® probes are a type of oligonucleotide probe designed for real-time PCR (qPCR) applications. They are used for the detection and quantification of specific nucleic acid sequences.

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Lab products found in correlation

Qiaamp dna blood mini kit, by Qiagen (2 mentions) Lightcycler 1.5 platform, by Roche (2 mentions) Maxwell 16 blood dna purification kit, by Promega (1 mentions) Lightcycler 480 real time pcr system, by Roche (1 mentions) Lightcycler faststart dna master hybprobe, by Roche (1 mentions) Magna pure lc dna isolation kit large volume, by Roche (1 mentions) Lightcycler faststart dna master hybprobe mix, by Roche (1 mentions)

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SimpleProbe (2 citations)

4 protocols using simpleprobe probes

Genotyping IL-33 Gene Variants

Cited 1 time

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The selection of genetic variants within the IL-33 gene was based on an analysis of the available literature and search results from the HapMap and NCBI dbSNP databases. Information of the predicted functional consequences of SNPs was obtained using the SNPinfo Web Server (34 (link)). The studied SNPs were characterized with minor allele frequencies above 10% (1000 Genomes Project) (35 (link)).
Peripheral venous blood from each subject was collected in ethylenediaminetetraacetic acid (EDTA) anticoagulant tubes. DNA isolation was carried out using a Maxwell 16 Blood DNA Purification Kit (Promega Corp., Madison, WI, USA). The IL-33 SNPs were detected by real-time PCR using LightCycler Technology with SimpleProbe probes (LightSNiP assays) designed by TIB MolBiol (Berlin, Germany). Genotyping was performed on a LightCycler 480 Real-Time PCR system (Roche Diagnostics, Rotkreuz, Switzerland) according to the manufacturer’s instructions.

Iwaszko M., Wielińska J., Świerkot J., Kolossa K., Sokolik R., Bugaj B., Chaszczewska-Markowska M., Jeka S, & Bogunia-Kubik K. (2021). IL-33 Gene Polymorphisms as Potential Biomarkers of Disease Susceptibility and Response to TNF Inhibitors in Rheumatoid Arthritis, Ankylosing Spondylitis, and Psoriatic Arthritis Patients. Frontiers in Immunology, 12, 631603.

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SNP Genotyping of TSLP and IL-33

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Genomic DNA was obtained from EDTA whole blood samples using a QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany). The samples of the 78 subjects were genotyped for specific SNPs, including TSLP rs11466750 and rs2289277, and IL-33 rs992969 and rs1888909. All SNPs were determined with real-time polymerase chain reaction (PCR) assays with subsequent melting curve analysis using SimpleProbe^® probes (TibMolbiol, Berlin, Germany), which were complementary to the wild-type sequences.
PCR was performed at a final volume of 10 µL containing 1 µL of DNA at a concentration of 15–60 ng/µL, 0.5 µL of reagent mix containing specific primers and SimpleProbes^® probesat optimized concentration, 0.8 µL of MgCl₂, and 1 µL of LightCycler^®FastStart DNA MasterHybProbe (Roche Applied Science, Mannheim, Germany). The reactions were performed on a Light Cycler 1.5 platform (Roche Applied Science, Mannheim, Germany). To test the quality of every step of the genotyping procedures, negative controls for each genotype were included in each reaction.

Połomska J., Sikorska-Szaflik H., Drabik-Chamerska A., Sozańska B, & Dębińska A. (2024). Exploring TSLP and IL-33 Serum Levels and Genetic Variants: Unveiling Their Limited Potential as Biomarkers for Mild Asthma in Children. Journal of Clinical Medicine, 13(9), 2542.

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CYP2C19 Genotyping Protocol

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As a part of the study procedures, a 7ml EDTA-blood sample was taken from all participants. For genotyping, genomic DNA was isolated from the biosamples using magnetic beads with the MagNA PureLC DNA Isolation Kit-Large Volume (Roche  3  Diagnostics GmbH., Mannheim, Germany). Genotypes for CYP2C19, rs12248560 (C>T) (CYP2C19*17) and rs42442085 (G>A) (CYP2C19*2), were determined by real-time PCR using SimpleProbe® probes (LightSNiP assays), designed by TIB Molbiol (Berlin, Germany). To this end, 50 ng DNA were amplified using Lightcycler® FastStart DNA Master HybProbe Mix (Roche, Germany) and the respective LightSNiP Assay. The following PCR protocol was applied: 10 min at 95°C, followed by 45 cycles for amplification: 10 s at 95°C, 10 s at 60°C, 15 s at 72°C. Melting curve analysis was performed to distinguish between the different genotypes. We used a goodness-of-fit c2 test to evaluate agreement with Hardy-Weinberg expectations. CYP2C19 diplotypes were assigned activity scores as shown in Table 1. If no mutation for rs12248560 and rs42442085was detected, wildtype carrier status was assumed.

Kirchheiner J., Scholl C., Bosch J.E., & Viviani R. (2020). Genetic polymorphism of CYP2C19 and subcortical variability in the human adult brain.

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Genotyping of Immune Regulation Genes

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The samples of the 115 subjects were genotyped for the CD14 C-159T, TLR4 +896 A/G and TLR2 A-16934T polymorphisms. Genomic DNA was obtained from ethylenediaminetetraacetic acid (EDTA) whole blood samples using a QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany). All SNPs were determined with realtime polymerase chain reaction (PCR) assays with subsequent melting curve analysis using SimpleProbe ® probes (TibMolbiol, Berlin, Germany) which were complementary to the wild-type sequences. Initial PCR amplifications and melting analyses were carried out with various oligonucleotide concentrations and ratios. Melting peaks were optimized when asymmetric PCR conditions (unequal amounts of primers) were used. The reactions were performed on a Light Cycler 1.5 platform (Roche Applied Science, Mannheim, Germany) as described in Table 1. For quality control of the genotyping procedures, positive and negative controls for each genotype were included in each reaction.

Dębińska A., Danielewicz H., Drabik-Chamerska A., Kalita D, & Boznański A. (2019). Genetic polymorphisms in pattern recognition receptors are associated with allergic diseases through gene-gene interactions. Advances in clinical and experimental medicine : official organ Wroclaw Medical University, 28(8).

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