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SALSA MLPA Probemix ME042 CIMP

CpG Island methylator phenotype (CIMP)

Region: Various

This product is for basic research and intended for experienced users only! Interpretation of results may be complicated, and MRC Holland may only be able to provide basic support.


General information
The SALSA MS-MLPA Probemix ME042 CIMP is a research use only (RUO) assay for the detection of aberrant methylation of one or more sequences in the promoter regions of the CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1 genes. This probemix can also be used to detect deletions/duplications in the promoter regions aforementioned genes, and the presence of the BRAF p.V600E (c.1799T>A) point mutation in a DNA sample.

CpG-islands are located in or near the promoter region or other regulatory regions of approximately 50% of human genes. Aberrant methylation of CpG-islands has been shown to be associated with transcriptional inactivation of genes in a wide spectrum of human cancers. The CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1 genes are frequently silenced by methylation in CpG Island Methylator Phenotype (CIMP) tumours, but are unmethylated in blood-derived DNA, with the exception of constitutional epimutations. In addition, DNA methylation analysis can indicate in some cases from which type of tissue the tumour was derived.

CIMP tumours are characterized by the hypermethylation of promoters mainly in tumour suppressor genes and DNA repair genes, which leads to genetic silencing and a loss of protein expression. CIMP is a typical feature for a subset of colorectal cancers, where also BRAF mutations are commonly found (Weisenberger et al. 2006). CIMP status was also identified in glioblastoma, gastric cancer, endometrial and breast cancers (reviewed in Weisenberger (2014)).

This SALSA MS-MLPA probemix is not CE/FDA registered for use in diagnostic procedures. Purchase of this product includes a limited license for research purposes.

Exon numbering

The exon numbering used in this ME042-C2 CIMP product description is the exon numbering from the sequence of NG_032024.1 for CACNA1G, of NM_004378.3 for CRABP1, of LRG_1031 for IGF2, of LRG_216 for MLH1, of NM_006161.3 for NEUROG1, of NM_001031680.2 for RUNX3 and of NM_003745.2 for SOCS1.
From product description version C2-03 onwards, the exon numbering from the MANE transcripts is used for CDKN2A.
Consequently, for CDKN2A, the exon numbering has been changed: NM_000077.5 (MANE Select) encoding p16INK4A and NM_058195.4 (MANE Plus Clinical) encoding p14ARF are used. Both NM_000077.5 and NM_058195.4 have distinct first exons (both numbered as exon 1) which contain the translation start codon, and share a common second exon, which is translated in different reading frames.
The exon numbering (LRG_11 for CDKN2A), used in previous versions of this product description, can be found in between brackets in the Table 2.
Please be aware that the MANE and LRG exon numbering do not always correspond, and MANE exon numbering used here may differ from literature.
The exon numbering of the NG/NM_ sequence that was used for determining a probe’s ligation site does not always correspond to the exon numbering obtained from the LRG sequences. As changes to the databases can occur after release of this product description, the NG/NM_ sequence and exon numbering may not be up-to-date.

Probemix content
The SALSA MS-MLPA Probemix ME042-C2 CIMP contains 49 (MS-)MLPA probes with amplification products between 124 and 492 nucleotides (nt). 31 MS-MLPA probes contain an HhaI recognition site and provide information on the methylation status of promoter regions of CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1 genes. All probes present will also give information on copy number changes in the analysed sample. In addition, 15 reference probes are included that are not affected by HhaI digestion and target relatively copy number stable regions in various cancer types. Also, two digestion control probes are included in this probemix indicating whether or not restriction endonuclease digestion in the MS-MLPA reaction was complete. Complete probe sequences and the identity of the genes detected by the reference probes are available online and in Table 3 (www.mrcholland.com).
This probemix contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and one chromosome Y-specific fragment. More information on how to interpret observations on these control fragments can be found in the MS-MLPA General Protocol and online at www.mrcholland.com.

SALSA Binning DNA SD029
The SD029 Binning DNA provided with this probemix can be used for binning of all probes including the BRAF p.V600E (c.1799T>A) mutation-specific probe (226 nt probe 08780-SP0039-L08904). SD029 Binning DNA is a mixture of genomic DNA from healthy individuals and plasmid DNA that contains the target sequence detected by the above mentioned probe. Inclusion of one reaction with 5 μl SD029 Binning DNA in initial MLPA experiments is essential as it can be used to aid in data binning of the peak pattern using Coffalyser.Net software. Furthermore, Binning DNA should be included in the experiment whenever changes have been applied to the set-up of the capillary electrophoresis device (e.g. when capillaries have been renewed). Binning DNA should never be used as a reference sample in the MLPA data analysis, neither should it be used in quantification of a mutation signal. It is strongly advised that all samples tested are extracted with the same method and derived from the same source of tissue. For further details, please consult the SD029 Binning DNA product description, available online: www.mrcholland.com. This product is for research use only (RUO).

Sample DNA
Sample DNA developed for this product:

Order Items

Probemix

Item no.
Description
Technology
Price
ME042-025R
SALSA MLPA Probemix ME042 CIMP – 25 rxn
€ 281.00
ME042-050R
SALSA MLPA Probemix ME042 CIMP – 50 rxn
€ 550.00
ME042-100R
SALSA MLPA Probemix ME042 CIMP – 100 rxn
€ 1075.00

Required Reagents (Sold Separately)

Item no.
Description
Technology
Price
EK1-FAM
SALSA MLPA Reagent Kit – 100 rxn – FAM (6 vials)
€ 341.00
EK1-Cy5
SALSA MLPA Reagent Kit – 100 rxn – Cy5 (6 vials)
€ 341.00
EK5-FAM
SALSA MLPA Reagent Kit – 500 rxn – FAM (5×6 vials)
€ 1571.00
EK5-Cy5
SALSA MLPA Reagent Kit – 500 rxn – Cy5 (5×6 vials)
€ 1571.00
EK20-FAM
SALSA MLPA Reagent Kit – 2000 rxn – FAM (5×6 vials)
€ 6037.00
SMR50
SALSA HhaI – 115 μl
€ 45.00

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