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> Chemicals & Drugs > Amino Acid > Hypoxanthine Phosphoribosyltransferase

Hypoxanthine Phosphoribosyltransferase

Hypoxanthine Phosphoribosyltransferase (HPRT) is a crucial enzyme invloved in the purine salvage pathway, catalyzing the conversion of hypoxanthine to inosine monophosphate.
It plays a vital role in maintaining cellular purine levels and is essential for proper cell function.
Mutations in the HPRT gene can lead to Lesch-Nyhan syndrome, a rare genetic disorder characterized by neurological and behavioral abnormalities.
Understanding HPRT's structure, function, and regulation is crucial for developing therapies targeting purine metabolism disorders.

Most cited protocols related to «Hypoxanthine Phosphoribosyltransferase»

1
Quantifying T. gondii Gene Expression
Cited 30 times
FHs 74 Int cells were mock-infected or infected with the GFP-RH strain of T. gondii at an MOI of 10 for 4 or 8 h. Total cellular RNA was extracted using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and 3 μg RNA was reverse-transcribed in a final volume of 20 μl using Superscript II reverse transcriptase (Invitrogen Life Technologies) as described by the manufacturer. qRT-PCR was performed using SYBR Premix Ex Taq II reagent (Takara Bio Inc., Dalian, China) as described previously [3 (link)]. Each reaction included 1 μl of cDNA (100 ng/μl), 10 μl of SYBR Premix Ex Taq II (2X), 0.8 μl of forward and reverse primers (10 μM), 0.4 μl of ROX reference Dye II and 7 μl of DNase-/RNase-free PCR water to a final volume of 20 μl. The primers used in this study are summarized in Additional file 1: Table S1. All reactions were performed with an ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA) under the following conditions: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. Relative gene expression levels were quantified based on the cycle threshold (Ct) values and were normalized to the reference gene hypoxanthine phosphoribosyltransferase 1 (HPRT1). Each sample was measured in triplicate, and the gene expression levels were calculated using the 2–ΔΔCt method.
Quan J.H., Huang R., Wang Z., Huang S., Choi I.W., Zhou Y., Lee Y.H, & Chu J.Q. (2018). P2X7 receptor mediates NLRP3-dependent IL-1β secretion and parasite proliferation in Toxoplasma gondii-infected human small intestinal epithelial cells. Parasites & Vectors, 11, 1.
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Publication 2018
Cells Deoxyribonuclease I DNA, Complementary Gene Expression Genes Hypoxanthine Phosphoribosyltransferase Oligonucleotide Primers Ribonuclease 7 RNA-Directed DNA Polymerase Strains trizol
2
Quantitative PCR Analysis of Cytokines and Enzymes
Cited 17 times
RNA was isolated from organs using the RNeasy Mini Kit (QIAGEN). mRNA was reverse transcribed and analyzed in triplicate assays by TaqMan PCR using a sequence detection system (ABI Prism 7700; Applied Biosystems), as described previously (Wolk et al., 2002 (link)). For detection of mouse IFN-γ, IL-12, IL-10, IL-6, IL-1β, IL-17A, IL-22, MMP-2, MMP-9, and IL-23p19, assays including double-fluorescent probes in combination with assays for the mouse housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) were purchased from Applied Biosystems or developed by us (IL-22). Expression levels were calculated relative to the HPRT expression.
Muñoz M., Heimesaat M.M., Danker K., Struck D., Lohmann U., Plickert R., Bereswill S., Fischer A., Dunay I.R., Wolk K., Loddenkemper C., Krell H.W., Libert C., Lund L.R., Frey O., Hölscher C., Iwakura Y., Ghilardi N., Ouyang W., Kamradt T., Sabat R, & Liesenfeld O. (2009). Interleukin (IL)-23 mediates Toxoplasma gondii–induced immunopathology in the gut via matrixmetalloproteinase-2 and IL-22 but independent of IL-17. The Journal of Experimental Medicine, 206(13), 3047-3059.
Publication 2009
Biological Assay Fluorescent Probes Genes, Housekeeping Hypoxanthine Phosphoribosyltransferase IFNG protein, mouse IL10 protein, human IL22 protein, human Interleukin-1 beta Interleukin-12 Interleukin-17A Interleukin-23 Subunit p19 MMP2 protein, human MMP9 protein, human Mus prisma RNA, Messenger
3
Quantifying Porcine mRNA Transcripts
Cited 12 times
Many mRNA sequences have already been identified in the pig. When genes were not described in this species, tBLASTn searches of the GenBank and PEDEblast EST databases, using known human and murine amino acid sequences, were performed. This methodology enables the identification of porcine expressed sequence tags (EST) corresponding to human and murine sequences (Tab. I). Then, primers (Tab. I) were designed using Clone Manager 9 (Scientific & Educational Software) and were purchased from Eurogentec (Liège, Belgium).
Quantitative real-time PCR (qPCR) was performed using cDNA synthesized as previously described [29 (link)]. Diluted cDNA (40×) was combined with primer/probe sets and IQ SYBR Green Supermix (Bio-Rad, Hercules, California, USA) according to the manufacturer’s recommendations. The qPCR conditions were 95 °C for 3 min, followed by 45 cycles with denaturation at 95 °C for 15 s, annealing temperature (Tab. I) for 30 s and elongation at 72 °C for 30 s. Real time assays were run on a Bio-Rad iCycler iQ. The specificity of the qPCR reactions was assessed by analyzing the melting curves of the products and size verification of the amplicons. To minimize sample variation, tissue samples of similar size and location and identical quantities of high quality RNA with no signs of degradation were used. Samples were normalized internally using simultaneously the average cycle threshold (Ct) of Hypoxanthine PhosphoRibosyl-Transferase 1 (HPRT-1), Ribosomal Protein L 19 (RPL-19) and Tata Box Binding Protein 1 (TBP-1) [33 (link)] as references in each sample to avoid any artifact of variation in the target gene. HPRT-1, RPL-19 and TBP-1 genes were selected as the reference genes because of their extremely low variation among samples. A standard curve was generated using diluted cDNA. The correlation coefficients of the standard curves were > 0.995 and the concentration of the test samples were calculated from the standard curves, according to the formula y = −M × Ct + B, where M is the slope of the curve, Ct the point during the exponential phase of amplification in which the fluorescent signal is first recorded as being statistically significant above background and B the y-axis intercept. All qPCR displayed efficiency between 90% and 110% according to the equation: qPCR efficiency = (10[−1/M] − 1) × 100. Expression data are expressed as relative values after Genex macro analysis (Bio-Rad) [40 ].
Meurens F., Berri M., Auray G., Melo S., Levast B., Virlogeux-Payant I., Chevaleyre C., Gerdts V, & Salmon H. (2008). Early immune response following Salmonella enterica subspecies enterica serovar Typhimurium infection in porcine jejunal gut loops. Veterinary Research, 40(1), 05.
Publication 2008
Amino Acid Sequence Biological Assay Clone Cells DNA, Complementary Epistropheus Expressed Sequence Tags Genes Homo sapiens Hypoxanthine Phosphoribosyltransferase Mus Oligonucleotide Primers Pigs Real-Time Polymerase Chain Reaction Ribosomal Proteins RNA, Messenger SYBR Green I TATA-Box Binding Protein Tissues
4
RNA Extraction and qPCR Analysis of Splicing Factors
Cited 8 times
Total RNA from fresh normal and tumour human and mouse tissue samples was extracted, followed by DNase treatment using the AllPrep DNA/RNA/Protein Mini Kit and the RNase-Free DNase set (Qiagen, #80004/#79254), respectively. Total RNA from glioblastoma cell lines was extracted with TRIzol® Reagent (ThermoFisher Scientific, #15596026). In all cases, total RNA concentration and purity were assessed by Nanodrop One Microvolume UV-Vis Spectrophotometer (ThermoFisher Scientific). For qPCR analyses, total RNA was retrotranscribed by using random hexamer primers and the RevertAid RT Reverse Transcription Kit (ThermoFisher Scientific, #K1691). Thermal profile and qPCR analysis to obtain absolute mRNA copy number/50 ng of sample of selected genes are reported elsewhere (Luque et al., 2013 (link), 2015 (link)).
As recently reported (Jimenez-Vacas et al., 2019b (link), 2020 (link)), a qPCR dynamic array based on microfluidic technology (Fluidigm, #BMK-M-48.48) was implemented to determine the simultaneous expression of 48 transcripts in HGA/glioblastoma samples compared to control samples using the Biomark System and the Fluidigm® Real-Time PCR Analysis Software v.3.0.2 and Data Collection Software v.3.1.2 (Fluidigm). Specific primers for human and mouse transcripts including components of the major spliceosome (n = 13), minor spliceosome (n = 4), associated splicing factors (n = 28), PDGFRB pathway-related genes and three housekeeping genes were specifically designed with the Primer3 software (Supplementary Tables 2–4). To control for variations in the efficiency of the retrotranscription reaction, mRNA copy numbers of the different transcripts analysed were adjusted by a normalization factor, calculated with the expression levels of three housekeeping genes [β-actin (ACTB), hypoxanthine guanine phosphoribosyl-transferase (HPRT), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; only for human samples) and peptidylprolyl isomerase-A (Cyclophilin A; only for mouse samples)] (Supplementary Tables 2 and 3) and the GeNorm v.3.3 software as previously reported (Luque et al., 2015 (link); Hormaechea-Agulla et al., 2017a (link); Jimenez-Vacas et al., 2019a (link)).
Fuentes-Fayos A.C., Vázquez-Borrego M.C., Jiménez-Vacas J.M., Bejarano L., Pedraza-Arévalo S., L.-López F., Blanco-Acevedo C., Sánchez-Sánchez R., Reyes O., Ventura S., Solivera J., Breunig J.J., Blasco M.A., Gahete M.D., Castaño J.P, & Luque R.M. (2020). Splicing machinery dysregulation drives glioblastoma development/aggressiveness: oncogenic role of SRSF3. Brain, 143(11), 3273-3293.
Publication 2020
Actins Cell Lines Cyclophilin A Deoxyribonuclease I Endoribonucleases factor A GAPDH protein, human Genes Genes, Housekeeping Glioblastoma Glyceraldehyde-3-Phosphate Dehydrogenases Homo sapiens Hypoxanthine Phosphoribosyltransferase Mus Neoplasms Oligonucleotide Primers Peptidylprolyl Isomerase Proteins Real-Time Polymerase Chain Reaction Reverse Transcription RNA, Messenger RNA Splicing Factors Spliceosomes Tissues trizol
5
Conditional Gene Modification in Toxoplasma
Cited 8 times
The parental strain used for encysting Toxoplasma was the type II PruΔhpt in which the endogenous gene for hypoxanthine xanthine guanine phosphoribosyl transferase (HPT) has been deleted. All strains were propagated in human foreskin fibroblasts (HFFs). The vector expressing the selectable HPT marker and the epitope-tagged rhoptry protein fused to Cre recombinase (pToxofilin-Cre) has been previously described [6] (link). In addition to the pToxofilin-Cre plasmid, a second plasmid containing the coding sequence for mCherry, flanked by the GRA2 promoter and 5′-UTR and the GRA2 3′-UTR, was co-transfected into the parasites. The parental strain for all non-encysting parasites was the type I strain RHΔhpt[39] (link). The generation of RH-Cre and the vector expressing HPT and a toxofilin:β-lactamase fusion have been previously described [20] (link). In addition to the pToxofilin-β-lactamase plasmid, a second plasmid containing the coding sequence for mCherry flanked by the GRA1 promoter and 5′-UTR and GRA2 3′-UTR, was co-transfected into the parasites. To generate mCherry+ parasites expressing the respective toxofilin fusion protein, the parental parasites were electroporated with the appropriate plasmids, linearized upstream of the relevant expression cassettes prior to electroporation. As previously described, the parasites were then subjected to several rounds of selection for expression of HPT using medium containing 25 µg/ml mycophenolic acid and 50 µg/ml xanthine before being cloned by limiting dilution [40] (link). Single cell clones that were HPT+ and mCherry+ and confirmed to express the appropriate toxofilin fusion protein were then tested for efficacy in causing Cre-mediated recombination in a Cre-reporter cell line and Cre-reporter mice [6] (link), or for the ability to cleave the substrate CCF2-AM [20] (link) to verify secretion of a functional toxofilin:Cre or toxofilin:β-lactamase fusion protein, respectively. For the encysting Pru-Cre strain, a control parasite strain was also selected that both expressed mCherry and had the selectable marker but, unlike the Pru-Cre strain, expressed a truncated form of the Cre fusion protein, as determined by western blot analyses (data not shown). This truncated fusion protein was inactive as infection of either the Cre-reporter cells or mice with this control strain did not result in Cre-mediated recombination (data not shown).
Koshy A.A., Dietrich H.K., Christian D.A., Melehani J.H., Shastri A.J., Hunter C.A, & Boothroyd J.C. (2012). Toxoplasma Co-opts Host Cells It Does Not Invade. PLoS Pathogens, 8(7), e1002825.
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Publication 2012
5' Untranslated Regions beta-Lactamase Cell Lines Cells Clone Cells Cloning Vectors Cre recombinase Electroporation Epitopes Fibroblasts Foreskin Genes Homo sapiens Hypoxanthine hypoxanthine-guanine-xanthine phosphoribosyltransferase Hypoxanthine Phosphoribosyltransferase Infection Mice, Laboratory Mycophenolic Acid Open Reading Frames Parasites Parent Plasmids Proteins Recombination, Genetic secretion Staphylococcal Protein A Strains Technique, Dilution Toxoplasma Western Blot Xanthine xanthine phosphoribosyltransferase

Most recents protocols related to «Hypoxanthine Phosphoribosyltransferase»

1
Generation and Maintenance of Haploid Human Embryonic Stem Cells
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Example 3

We generated and analyzed a collection of 14 early-passage (passage ≤9) human pES cell lines for the persistence of haploid cells. All cell lines originated from activated oocytes displaying second polar body extrusion and a single pronucleus. We initially utilized chromosome counting by metaphase spreading and G-banding as a method for unambiguous and quantitative discovery of rare haploid nuclei. Among ten individual pES cell lines, a low proportion of haploid metaphases was found exclusively in a single cell line, pES10 (1.3%, Table 1B). We also used viable FACS with Hoechst 33342 staining, aiming to isolate cells with a DNA content corresponding to less than two chromosomal copies (2c) from four additional lines, leading to the successful enrichment of haploid cells from a second cell line, pES12 (Table 2).

Two individual haploid-enriched ES cell lines were established from both pES10 and pES12 (hereafter referred to as h-pES10 and h-pES12) within five to six rounds of 1c-cell FACS enrichment and expansion (FIG. 1C (pES10), FIG. 5A (pES12)). These cell lines were grown in standard culture conditions for over 30 passages while including cells with a normal haploid karyotype (FIG. 1D, FIG. 5B). However, since diploidization occurred at a rate of 3-9% of the cells per day (FIG. 1E), cell sorting at every three to four passages was required for maintenance and analysis of haploid cells. Further, visualization of ploidy in adherent conditions was enabled by DNA fluorescence in situ hybridization (FISH) (FIG. 1F, FIG. 5c) and quantification of centromere protein foci (FIG. 1G, FIG. 5D; FIG. 6). In addition to their intact karyotype, haploid ES cells did not harbor significant copy number variations (CNVs) relative to their unsorted diploid counterparts (FIG. 5E). Importantly, we did not observe common duplications of specific regions in the two cell lines that would result in pseudo-diploidy. Therefore, genome integrity was preserved throughout haploid-cell isolation and maintenance. As expected, single nucleotide polymorphism (SNP) array analysis demonstrated complete homozygosity of diploid pES10 and pES12 cells across all chromosomes.

Both h-pES10 and h-pES12 exhibited classical human pluripotent stem cell features, including typical colony morphology and alkaline phosphatase activity (FIG. 2A, FIG. 2B). Single haploid ES cells expressed various hallmark pluripotency markers (NANOG, OCT4, SOX2, SSEA4 and TRA1-60), as confirmed in essentially pure haploid cultures by centromere foci quantification (>95% haploids) (FIG. 2C, FIG. 7). Notably, selective flow cytometry enabled to validate the expression of two human ES-cell-specific cell surface markers (TRA-1-60 and CLDN618) in single haploid cells (FIG. 2D). Moreover, sorted haploid and diploid ES cells showed highly similar transcriptional and epigenetic signatures of pluripotency genes (FIG. 2E, FIG. 2F). Since the haploid ES cells were derived as parthenotes, they featured distinct transcriptional and epigenetic profiles of maternal imprinting, owing to the absence of paternally-inherited alleles (FIG. 8).

Haploid cells are valuable for loss-of-function genetic screening because phenotypically-selectable mutants can be identified upon disruption of a single allele. To demonstrate the applicability of this principle in haploid human ES cells, we generated a genome-wide mutant library using a piggyBac transposon gene trap system that targets transcriptionally active loci (FIG. 2G, FIG. 8E), and screened for resistance to the purine analog 6-thioguanine (6-TG). Out of six isolated and analyzed 6-TG-resistant colonies, three harbored a gene trap insertion localizing to the nucleoside diphosphate linked moiety X-type motif 5 (NUDT5) autosomal gene (FIG. 2H). NUDT5 disruption was recently confirmed to confer 6-TG resistance in human cells,51 by acting upstream to the production of 5-phospho-D-ribose-1-pyrophosphate (PRPP), which serves as a phosphoribosyl donor in the hypoxanthine phosphoribosyltransferase 1 (HPRT1)-mediated conversion of 6-TG to thioguanosine monophosphate (TGMP) (FIG. 2I). Detection of a loss-of-function phenotype due to an autosomal mutation validates that genetic screening is feasible in haploid human ES cells.

US11859232B2. Screening for chemotherapy resistance in human haploid cells (2024-01-02). YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM LTD. [IL]. Inventors: Nissim Benvenisty [IL].
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Patent 2024
Alkaline Phosphatase Alleles Cell Lines Cell Nucleus Cells Cell Separation Centromere Chromosomes Copy Number Polymorphism Diphosphates Diploid Cell Diploidy Embryonic Stem Cells Flow Cytometry Fluorescent in Situ Hybridization Genes Genes, vif Genitalia Genome Genomic Library Haploid Cell HOE 33342 Homo sapiens Homozygote Human Embryonic Stem Cells Hypoxanthine Phosphoribosyltransferase isolation Jumping Genes Karyotype Metaphase Mothers Mutation Nucleosides Oocytes Phenotype Pluripotent Stem Cells Polar Bodies POU5F1 protein, human Proteins purine Ribose Single Nucleotide Polymorphism SOX2 protein, human stage-specific embryonic antigen-4 Tissue Donors Transcription, Genetic
2
Ileal and Liver RNA Extraction and Analysis
DNA-free RNA was obtained from ileal or liver tissue using the RNeasy Mini Kit (QIAGEN) with Dnase treatment. 0.5 μg total RNA was reverse transcribed using SuperScript VILO MasterMix (Life Technologies) by thermal cycled at 25 °C for 10 min, 42 °C for 60 min, and 85 °C for 5 min using Mastercycler EP (Eppendorf). Quantitative PCR was performed using Roche LightCycler 96 (Roche) with fluorescence-labeled locked nucleic acid (LNA) hydrolysis probes (Roche) from the Universal Probe Library (UPL) following the manufacturer’s protocol. Gene expression was normalized to hypoxanthine guanine phosphoribosyl transferase-1 (Hprt1). The primer sequences are listed in Supplementary Table 2.
Nakamura S., Nakamura K., Yokoi Y., Shimizu Y., Ohira S., Hagiwara M., Song Z., Gan L., Aizawa T., Hashimoto D., Teshima T., Ouellette A.J, & Ayabe T. (2023). Decreased Paneth cell α-defensins promote fibrosis in a choline-deficient L-amino acid-defined high-fat diet-induced mouse model of nonalcoholic steatohepatitis via disrupting intestinal microbiota. Scientific Reports, 13, 3953.
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Publication 2023
Deoxyribonucleases DNA Library Fluorescence Gene Expression Hydrolysis Hypoxanthine Phosphoribosyltransferase Ileum Liver locked nucleic acid Nucleic Acid Probes Oligonucleotide Primers Tissues
3
Real-time PCR Analysis of Tissue Gene Expression
Real-time PCR was performed as previously described (51 (link)). Briefly, RNA was extracted from whole tissue by RNeasy mini kit (Qiagen, Venlo, Netherlands). Complementary DNA was reverse transcribed from 1 µg total RNA with Moloney murine leukemia virus reverse transcriptase (Thermo Fisher Scientific) using random hexamer oligonucleotides for priming (Thermo Fisher Scientific). The amplification was performed with a Biorad CFX-Connect Real-time-System (Thermo Fisher Scientific) using the SYBR Green (Eurogentec, Seraing, Belgium) or TaqMan (Thermo Fisher Scientific) detection system. Data were analyzed using the software supplied with the Sequence Detector (Life Technologies). The mRNA content was normalized to the hypoxanthine-guanine phosphoribosyltransferase (Hprt) mRNA for mouse genes. Gene expression was quantified using the ΔΔCt method.
Bénard A., Hansen F.J., Uhle F., Klösch B., Czubayko F., Mittelstädt A., Jacobsen A., David P., Podolska M.J., Anthuber A., Swierzy I., Schaack D., Mühl-Zürbes P., Steinkasserer A., Weyand M., Weigand M.A., Brenner T., Krautz C., Grützmann R, & Weber G.F. (2023). Interleukin-3 protects against viral pneumonia in sepsis by enhancing plasmacytoid dendritic cell recruitment into the lungs and T cell priming. Frontiers in Immunology, 14, 1140630.
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Publication 2023
DNA, Complementary Gene Expression Genes Hypoxanthine Phosphoribosyltransferase Moloney Leukemia Virus Mus Oligonucleotides Real-Time Polymerase Chain Reaction RNA, Messenger RNA-Directed DNA Polymerase SYBR Green I Tissues
4
Quantitative real-time PCR for cannabinoid receptors
One-step quantitative real-time PCR was performed using the QuantiNova Multiplex RT-PCR kit (Qiagen GmbH, Hilden, Germany) and Taqman gene expression assays (Life Technologies, Carlsbad, CA, USA). Commercially available rat Taqman probes were used for cannabinoid receptor 1 (CB1R, cnr1 gene) and fatty acid amide hydrolase (FAAH) as target genes (Rn00562880_m1 for cnr1 and Rn00577086_m1 for faah) and hypoxanthine guanine phosphoribosyl transferase (HPRT) as reference gene (Rn01527840_m1). The relative gene expression was calculated using the delta-delta Ct method (Livak and Schmittgen, 2001 (link)) after normalization using the reference gene. All samples were run in 2 independent experimental replicates, and in triplicates within one gene assay.
Demaili A., Portugalov A., Dudai M., Maroun M., Akirav I., Braun K, & Bock J. (2023). Epigenetic (re)programming of gene expression changes of CB1R and FAAH in the medial prefrontal cortex in response to early life and adolescence stress exposure. Frontiers in Cellular Neuroscience, 17, 1129946.
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Publication 2023
Biological Assay fatty acid amide hydrolase Gene Expression Genes Hypoxanthine Phosphoribosyltransferase Real-Time Polymerase Chain Reaction Receptor, Cannabinoid Reverse Transcriptase Polymerase Chain Reaction
5
RNA Isolation and qPCR Analysis of Bone Markers
RNA isolation from the tibiae from experiment A was performed by pulverizing the diaphysis with a freezer mill in liquid nitrogen (Spex Certiprep 6750 FreezerMill, Spex Certiprep, Metuchen, NJ, USA). RNA was extracted from bone powder with Trizol for 1 hour at 37°C and re-extracted once with phenol and once with chloroform. Next, the samples were extracted again with Trizol in accordance with the manufacturer’s instructions (Invitrogen, Waltham, MA, USA). RNA extracts were incubated with DNAse to eliminate DNA contamination and stored at –80°C. The absorption at 260 and 280 nm was measured to determine the amount of RNA. A total of 100 ng of total RNA was transcribed into cDNA in a 20 μl mixture containing M-MLV Reverse Transcriptase (Promega, Fitchburg, WI, USA). All RNA samples were assayed in triplicate. Quantitative PCR was performed in a BioRad iCycler Real-time PCR system with three μl of each cDNA sample, 300 nM of each primer and SYBR Green Supermix (BioRad, Hercules, CA, USA) in a total volume of 25 μl. Table 1 lists the primer details of the genes of interest, which were Fgf23, Mepe, Dmp1, Phex, Cyp27b1 and Vdr and the housekeeping genes, which were hypoxanthine guanine phosphoribosyl transferase (Hprt) and porphobilinogen deaminase (Pbgd). After the PCR run a melting curve was conducted from 50°C to 95°C to check the specificity of the reactions. Mean Ct-values of each RNA sample assayed in triplicate were used. Gene expression for the genes of interest was normalized for the housekeeping genes ΔCt = (Ctgene of interest−Cthousekeeping gene) and expressed as fold difference from the average of the housekeeping genes (2-ΔCt) [28 (link)].
Nepal A.K., van Essen H.W., Reijnders C.M., Lips P, & Bravenboer N. (2023). Mechanical loading modulates phosphate related genes in rat bone. PLOS ONE, 18(3), e0282678.
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Publication 2023
Bones Chloroform Deoxyribonuclease I Diaphyses DMP1 protein, human DNA, Complementary DNA Contamination FGF23 protein, human Gene Expression Genes Genes, Housekeeping Hydroxymethylbilane Synthase Hypoxanthine Phosphoribosyltransferase isolation Nitrogen Oligonucleotide Primers Phenol Powder Promega RNA-Directed DNA Polymerase SYBR Green I Tibia trizol

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1
Rneasy mini kit by Qiagen
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The RNeasy Mini Kit is a laboratory equipment designed for the purification of total RNA from a variety of sample types, including animal cells, tissues, and other biological materials. The kit utilizes a silica-based membrane technology to selectively bind and isolate RNA molecules, allowing for efficient extraction and recovery of high-quality RNA.
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The LightCycler 480 is a real-time PCR instrument designed for quantitative nucleic acid analysis. It features a 96-well format and uses high-performance optics and detection technology to provide accurate and reliable results. The core function of the LightCycler 480 is to facilitate real-time PCR experiments through thermal cycling, fluorescence detection, and data analysis.
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More about "Hypoxanthine Phosphoribosyltransferase"

Hypoxanthine Phosphoribosyltransferase (HPRT) is a crucial enzyme involved in the purine salvage pathway, catalyzing the conversion of hypoxanthine to inosine monophosphate.
It plays a vital role in maintaining cellular purine levels and is essential for proper cell function.
Mutations in the HPRT gene can lead to Lesch-Nyhan syndrome, a rare genetic disorder characterized by neurological and behavioral abnormalities.
Understanding the structure, function, and regulation of HPRT is crucial for developing therapies targeting purine metabolism disorders.
Researchers can utilize various molecular biology techniques to study HPRT, such as RNA extraction with RNeasy Mini Kit or TRIzol reagent, cDNA synthesis with High-Capacity cDNA Reverse Transcription Kit or IScript cDNA synthesis kit, and real-time PCR analysis with StepOnePlus Real-Time PCR System, RNeasy kit, TaqMan Gene Expression Assays, LightCycler 480, or QuantiTect Reverse Transcription Kit.
Optimizing your HPRT research can be enhanced by using AI-driven platforms like PubCompare.ai, which can help you locate the best protocols from literature, pre-prints, and patents, ensuring reproducibility and accuracy.
With PubCompare.ai's powerful comparisons, you can experience enhanced research and gain deeper insights into the role of HPRT in purine metabolism and related disorders.