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S7810 CpG WIZ® RB1 - Methylation specific PCR assay

MSP, performed using the CpGenome DNA Modification Kit & the CpG WIZ RB1 Amplification Kit, permits sensitive detection of altered DNA.

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S7810
25 assays  
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      Overview

      Replacement Information
      Description
      Catalogue NumberS7810
      Brand Family Chemicon®
      Trade Name
      • CpG Wiz
      • Chemicon
      DescriptionCpG WIZ® RB1 - Methylation specific PCR assay
      OverviewMSP, performed using the CpGenome™ DNA Modification Kit and the CpG WIZ® RB1 Amplification Kit, permits sensitive detection of altered DNA. Because this is a PCR-based assay, it is extremely sensitive, facilitating the detection of low numbers of methylated alleles and the study of samples containing small amounts of DNA. MSP also allows examination of all CpG sites, not just those within sequences recognized by methylation sensitive restriction enzymes. Increasing the number of such sites which can be assessed allows rapid, fine mapping of methylation patterns throughout CpG regions. In addition, the bisulfite modification is ideally suited for analysis of CpG islands since it converts the majority of cytosines to uracils, making a region of the genome which is CG-rich less difficult to amplify by PCR.



      Methylation-specific PCR (MSP) employs an initial bisulfite reaction to modify the DNA, followed by a "hot start" PCR amplification with specific primers designed to distinguish methylated DNA from unmethylated DNA. As shown in Figure 1, in the bisulfite reaction, all unmethylated cytosines are converted to uracils while 5-methylcytosines remain unaltered. Thus, the sequence of the treated DNA will differ if the DNA is originally methylated vs. unmethylated. Primers contained in the CpG WIZ® RB1 Amplification Kit are designed to specifically amplify each of the sequences based upon these chemically-induced differences. If the sample DNA was originally unmethylated, a product will be generated after PCR using the U primer set. Conversely, a product will be generated using the M primer set if the sample was originally methylated.
      Materials Required but Not DeliveredEquipment and Supplies

      a. Microcentrifuge tubes for PCR amplification

      b. Aerosol-resistant pipette tips

      c. Thermocycler

      d. Gel electrophoresis apparatus (vertical or horizontal)

      e. Power Supply

      f. 302 nm UV transilluminator, camera and film

      Reagents

      a 2.5 mM dNTP mix (2.5 mM of each nucleotide)

      b. "Hot start" Taq polymerase

      c. "Hot start" PCR reagents (see Sec. II. Protocols).

      d. Reagents for gel electrophoresis (1X TBE and 2% agarose, 10% acrylamide, or suitable high resolution agarose)

      e. DNA markers (size range 100-300 bp)

      f. Ethidium bromide (10 mg/mL)

      g. Gel-loading solution / Loading Dye

      h. Bisulfite Modified DNA (CpGenome™ DNA Modification Kit, S7820)
      Background InformationMethylation of cytosines located 5' to guanosine is known to have a profound effect on the expression of several eukaryotic genes (1). In normal cells, methylation occurs predominantly in CG-poor regions, while CG-rich areas, called CpG islands, remain unmethylated. Aberrant methylation of normally unmethylated CpG islands has been documented as a relatively frequent event in immortalized and transformed cells (2) and has been associated with transcriptional inactivation of defined tumor suppressor genes in human cancers (3, 4). The retinoblastoma 1 (RB1) gene exhibits characteristic hypermethylation im many maligninant tumors.

      Previously developed methods to determine the methylation status of cytosine include digestion with methylation sensitive restriction enzymes and genomic DNA sequencing. Both techniques have limitations: restriction enzymes can only detect methylation sites within their recognition sequence and sequencing is time consuming. Increasing the detection sensitivity of CpG island methylation has the potential to define tumor suppressor gene function and provides a new strategy for early tumor detection.

      Methylation-specific PCR (MSP) is a new technology for sensitive detection of abnormal gene methylation utilizing small amounts of DNA (5). This process employs an initial bisulfite reaction to modify the DNA, followed by PCR amplification with specific primers designed to distinguish methylated from unmethylated DNA. The CpGenome™ DNA Modification Kit (S7820) contains the reagents necessary to perform the initial bisulfite reactions, while the CpG WIZ® RB1 Amplification Kit contains the reagents required for the PCR amplification reactions.
      References
      Product Information
      Components
      • The components of the CpG WIZ® RB1 Amplification Kit include those required for PCR amplification after bisulfite modification of DNA samples. Sufficient reagents are provided to analyze 25 samples with appropriate controls.
      • U Primer Set7.5 μM each primer (25X) 35 μL (neutral cap) 90552 -15°C to -25°C
      • M Primer Set7.5 μM each primer (25X) 35 μL (red cap) 90553 -15°C to -25°C
      • W Primer Set7.5 μM each primer (25X) 35 μL (green cap) 90554 -15°C to -25°C
      • U control DNA0.1 μg/μL 50 μL (white cap) 90393 -15°C to -25°C
      • M control DNA0.1 μg/μL 50 μL (red cap) 90394 -15°C to -25°C
      • W control DNA0.05 μg/μL 50 μL (green cap) 90395 -15°C to -25°C
      • Universal 10X PCR Buffer 265 μL (blue cap) 90396 -15°C to -25°C
      Quality LevelMQ100
      Applications
      ApplicationMSP, performed using the CpGenome DNA Modification Kit & the CpG WIZ RB1 Amplification Kit, permits sensitive detection of altered DNA.
      Key Applications
      • PCR
      Biological Information
      Species Reactivity
      • Human
      Entrez Gene Number
      Entrez Gene SummaryRetinoblastoma (RB) is an embryonic malignant neoplasm of retinal origin. It almost always presents in early childhood and is often bilateral. Spontaneous regression ('cure') occurs in some cases. The retinoblastoma gene RB was the first tumor suppressor gene cloned, and is a negative regulator of the cell cycle through its ability to bind the transcription factor E2F (MIM 189971) and repress transcription of genes required for S phase (Hanahan and Weinberg, 2000 [PubMed 10647931]).[supplied by OMIM]
      Gene Symbol
      • RB1
      • P105-RB
      • retinoblastoma-1
      • RB
      • PP110
      • OSRC
      UniProt Number
      UniProt SummaryFUNCTION: SwissProt: P06400 # Key regulator of entry into cell division that acts as a tumor suppressor. Directly involved in heterochromatin formation by maintaining overall chromatin structure and, in particular, that of constitutive heterochromatin by stabilizing histone methylation. Recruits and targets histone methyltransferases SUV39H1, SUV420H1 and SUV420H2, leading to epigenetic transcriptional repression. Controls histone H4 'Lys-20' trimethylation. Also acts as a transcription repressor of E2F target genes by recruiting chromatin-modifying enzymes to promoters. Inhibits the intrinsic kinase activity of TAF1. Forms a complex with adenovirus E1A and with SV40 large T antigen. May bind and modulate functionally certain cellular proteins with which T and E1A compete for pocket binding.
      SIZE: 928 amino acids; 106159 Da
      SUBUNIT: Interacts preferentially with transcription factor E2F1. The unphosphorylated form interacts with ARID3B, JARID1A, SUV39H1, MJD2A/JHDM3A and THOC1. Interacts with the N-terminal domain of TAF1. Interacts with AATF, DNMT1, LIN9, LMNA, SUV420H1, SUV420H2, PELP1 and TMPO-alpha. May interact with KNTC2. Interacts with EID1 and ZUBR1. Interacts with ARID4A and JARID1B.
      SUBCELLULAR LOCATION: Nucleus.
      TISSUE SPECIFICITY: Expressed in the retina.
      PTM: Phosphorylated from S to M phase of the cell cycle and is dephosphorylated in G1. T, but not E1A, binds only to the unphosphorylated form.
      DISEASE: SwissProt: P06400 # Defects in RB1 are the cause of childhood cancer retinoblastoma (RB) [MIM:180200]. RB is a congenital malignant tumor that arises from the nuclear layers of the retina. It occurs in about 1:20'000 live births and represents about 2% of childhood malignancies. It is bilateral in about 30% of cases. Although most RB appear sporadically, about 20% are transmitted as an autosomal dominant trait with incomplete penetrance. The diagnosis is usually made before the age of 2 years when strabismus or a gray to yellow reflex from pupil (cat eye) is investigated. & Defects in RB1 are a cause of bladder cancer [MIM:109800]. & Defects in RB1 are a cause of osteogenic sarcoma [MIM:259500].
      SIMILARITY: SwissProt: P06400 ## Belongs to the retinoblastoma protein (RB) family.
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Usage Statement
      • Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
      Storage and Shipping Information
      Packaging Information
      Material Size25 assays
      Transport Information
      Supplemental Information
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      S7810 04053252314759