SALSA MLPA Probemix P014-B1 Chromosome 8 Lot B1-0916 and B1-0713. Copy number changes of the human chromosome 8 are common in many types of tumours. In most cases, losses of 8p sequences and gains of 8q sequences are detected. This probemix is designed to have probes evenly spread over chromosome 8 and to contain probes for tumour suppressors (e.g. MTUS, LZTS1), DNA damage response genes (e.g. MCPH1) and cell cycle genes (e.g. MYC). For a complete list of chromosome 8 probes in this product, see Table 2a. This probemix contains 44 probes for different genes on chromosome 8 that are suggested to be involved in human tumours. In addition, 12 reference probes have been included in this probemix, detecting 12 different autosomal chromosomal locations, which are relatively silent in most tumour types. However, it should be noticed that tumour karyotypes can harbour multiple numerical and structural aberrations, which can complicate interpretation of these reference probes. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the above mentioned chromosomal regions in a DNA sample. Heterozygous deletions of recognition sequences should give a 35-50% reduced relative peak height of the amplification product of that probe. Note that a mutation or polymorphism in the sequence detected by a probe can also cause a reduction in relative peak height, even when not located exactly on the ligation site! In addition, some probe signals are more sensitive to sample purity and small changes in experimental conditions. Therefore, deletions and duplications detected by MLPA should always be confirmed by other methods. Not all deletions and duplications detected by MLPA will be pathogenic; users should always verify the latest scientific literature when interpreting their findings. We have no information on what percentage of defects in these genes is caused by deletions/duplications of complete exons. Finally, note that most defects in these genes are expected to be small (point) mutations, which will not be detected by this SALSA test. This SALSA MS- probemix is not CE/FDA registered for use in diagnostic procedures. Purchase of this product includes a limited license for research purposes. The use of a SALSA probemix and reagents requires a thermocycler with heated lid and sequence type electrophoresis equipment. Different fluorescent PCR primers are available. The MLPA technique has been first described in Nucleic Acids Research 30, e57 (2002). Related SALSA probemixes P175 Tumour gain: Contains probes for the following genes: MDM4, MYCN-ALK, PDGFRA, KIT, KDR, DHFR, EGFR, MET, SMO, BRAF/BRAF V600E mutation, FGFR1, MYC, ABL1, RET, CCND1, CCND2, CDK4, MDM2, AURKB, ERBB2-TOP2A, AURKA and AR. P294 Tumour loss: Contains probes for the following genes and chromosomal regions: 1p36, VHL, FHIT, APC, CDKN2A-CDKN2B, PTCH, TSC1, PTEN, WT1, BRCA2, RB1-MIR15, TSC2, TP53, NF1, BRCA1, SMAD4, STK11, FKBP8, SMARCB1 and FAM123B. References for SALSA P014 Chromosome 8 mix Yong ZWE et al (2014) Genetic alterations of chromosome 8 genes in oral cancer. Sci Rep. 4:6073. Passariello A et al (2013) Constitutional 11q14-q22 chromosome deletion syndrome in a child with neuroblastoma MYCN single copy. Eur J Med Genet. 56:626-34. Rumiato E et al (2011) DNA copy number profile discriminates between oesophageal adenocarcinoma and squamous cell carcinoma and represents an independent prognostic parameter in esophageal adenocarcinoma. Cancer Lett. 310:84-93. Kastler S et al (2010) POU5F1P1, a putative cancer susceptibility gene, is overexpressed in prostatic carcinoma. Prostate. 70:666-74. Bremmer JF (2005) A noninvasive genetic screening test to detect oral preneoplastic lesions. Lab Invest. 85:1481-8. Buffart TE et al (2005) DNA copy number changes at 8q11-24 in metastasized colorectal cancer. Cell Oncol. 27:57-65. SALSA probemix P014 Chromosome 8 Page 1 of 6
More information Website : www.mlpa.com E-mail : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands Data analysis The P014-B1 Chromosome 8 probemix contains 56 MLPA probes with amplification products between 123 and 504 nt. In addition, it contains 9 control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at 64-70-76-82 nt, three DNA Denaturation control fragments (D-fragments) at 88-92-96 nt, one X-fragment at 100 nt and one Y-fragment at 105 nt. More information on how to interpret observations on these control fragments can be found in the MLPA protocol. Data generated by this probemix should be normalised with a more robust method, as the target sites of the reference probes maybe gained or lost. (1) Intra-sample normalisation should be performed by dividing the signal of each target-specific probe by the signal of every single reference probe in that sample, thus creating as many ratios per target-specific probe as there are reference probes. Subsequently, the median of all these produced ratios per probe should be taken; this is the probe s Normalisation Constant. (2) Secondly, inter-sample comparison should be performed by dividing the Normalisation Constant of each probe in a given sample by the average Normalisation Constant of that probe in all the reference samples. Data normalisation should be performed within one experiment. Always use sample and reference DNA extracted with the same method and derived from the same source of tissue. Confirmation of deletions, duplications and amplifications can be done by e.g. Southern blotting, long range PCR, qpcr, FISH. Note that Coffalyser, the MLPA analysis tool developed at, can be downloaded free of charge from our website www.mlpa.com. Warning: MLPA analysis on tumour samples provides information on the average situation in the cells from which the DNA sample was purified. Gains or losses of genomic regions or genes may not be detected if the percentage of tumour cells is low. Furthermore, although reference probes are located in silent regions that are not frequently altered in copy number in most tumour types, there is always a possibility that one or more reference probes do show a copy number alteration in a sample. Normal copy number variation in healthy individuals is described in the database of genomic variants: http://dgv.tcag.ca/dgv/app/home. When in doubt, users should always verify the latest updates of the database and scientific literature when interpreting their findings. This probemix was developed at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA probemix P014 Chromosome 8 Page 2 of 6
Table 1. SALSA MLPA P014-B1 Chromosome 8 probemix Length Chromosomal position SALSA MLPA probe (nt) Reference Chr. 8p Chr. 8q MV Location (hg18) 64-70-76-82 Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA 88-92-96 D-fragments: Low signal of 88 or 96 nt fragment indicates incomplete denaturation 100 X-fragment: Specific for the X chromosome 105 Y-fragment: Specific for the Y chromosome 123 Reference probe 18709-L13645 5q31 05-132.037613 130 Reference probe 16316-L18705 3q21 03-129.999518 135 RIPK2 probe 17619-L25271 8q21.3 08-090.846711 139 PPP2R2A probe 18548-L25326 8p21.2 08-026.283646 145 GATA4 probe 07641-L20557 8p23.1 08-011.650002 150 Reference probe 03797-L25270 21q22 21-037.919598 154 LPL probe 11721-L21358 8p21.3 08-019.861015 160 MYC probe 00580-L22108 8q24.21 08-128.822148 166 «KLF10 probe 19026-L24836 8q22.3 08-103.733686 172 NCOA2 probe 09940-L10399 8q13.3 08-071.238280 178 MYBL1 probe 07915-L12249 8q13.1 08-067.641448 184 Reference probe 06587-L06145 2q24 02-165.939588 189 MYC probe 14869-L21679 8q24.21 08-128.822044 196 TCEB1 probe 18147-L22651 8q21.11 08-075.030773 202 TPD52 probe 01116-L00620 8q21.13 08-081.112844 211 «RECQL4 probe 01036-L00609 8q24.3 08-145.709833 217 NEFL probe 14108-L25272 8p21.2 08-024.866185 223 Reference probe 08064-L25273 9p13 09-034.480008 229 SLA probe 00488-L25274 8q24.22 08-134.120065 235 MCPH1 probe 15108-L25275 8p23.1 08-006.276358 241 TNFRSF10B probe 17475-L21781 8p21.3 08-022.941980 246 MOS probe 01037-L00621 8q12.1 08-057.188797 254 PTP4A3 probe 17603-SP0531-L25332 8q24.3 08-142.506804 261 «ZNF703 probe 17595-L25667 8p12 08-037.675173 266 Reference probe 04483-L03872 1p34 01-043.169314 274 E2F5 probe 01039-L00617 8q21.2 08-086.306924 279 CHD7 probe 06759-L25327 8q12.2 08-061.925685 284 RNF139 probe 04222-L16047 8q24.13 08-125.556613 292 MTDH probe 04151-L23263 8q22.1 08-098.742494 299 ASAP1 probe 16241-L18499 8q24.21 08-131.136182 305 Reference probe 07022-L23200 14q11 14-020.849856 312 LZTS1 probe 02552-L25663 8p21.3 08-020.151894 319 WHSC1L1 probe 19027-L25662 8p12 08-038.253030 325 PTK2 probe 01042-L25661 8q24.3 08-141.781923 333 PRKDC probe 05757-L25660 8q11.21 08-049.028942 338 RRM2B probe 11590-L15225 8q22.3 08-103.306313 346 EXT1 probe 03080-L02480 8q24.11 08-118.881023 353 NKX3-1 probe 17627-L25330 8p21.2 08-023.594273 361 Reference probe 06708-L25279 10p11 10-038.300693 368 NRG1 probe 19028-L25331 8p12 08-032.705041 373 FGFR1 probe 01046-L00624 8p12 08-038.434093 382 KHDRBS3 probe 01047-L00622 8q24.23 08-136.630197 391 KCNQ3 probe 06606-L06164 8q24.22 08-133.213617 400 EBAG9 probe 19029-L24839 8q23.2 08-110.632204 409 Reference probe 10681-L11263 6p12 06-051.932696 418 NDRG1 probe 19030-L25658 8q24.22 08-134.340582 425 DLGAP2 probe 06382-L05848 8p23.3 08-001.568441 432 EIF3H probe 01108-L25659 8q24.11 08-117.726436 439 Reference probe 16538-L21570 15q15 15-042.671892 445 PRDM14 probe 12002-L12830 8q13.3 08-071.130073 454 MTUS1 probe 19219-L25269 8p22 08-017.702395 463 WISP1 probe 19031-L24841 8q24.22 08-134.309404 472 * RAD54B probe 19032-L24842 8q22.1 08-095.492631 481 RHOBTB2 probe 19033-L24843 8p21.3 08-022.931348 SALSA probemix P014 Chromosome 8 Page 3 of 6
Length Chromosomal position SALSA MLPA probe (nt) Reference Chr. 8p Chr. 8q MV Location (hg18) 490 Reference probe 14909-L17529 18p11 18-013.724478 504 Reference probe 09870-L19465 2p15 02-061.126361 «This probe is located within, or close to, very strong CpG islands. A low signal of this probe can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. Note: Please notify us of any mistakes: info@mlpa.com. The identity of the genes detected by the reference probes is available in Table 2b. Table 2a. Chromosome 8 probes arranged according to chromosomal location Length SALSA MLPA Partial sequence Chromosomal Distance to Gene (nt) probe (24 nt adjacent to ligation site) position next probe 425 06382-L05848 DLGAP2 GTGAAGTTGAAT-CTCAGGCCATGG 8p23.3 4708 kb 235 15108-L25275 MCPH1 GCAAATACTCAT-TAGACTACCTTA 8p23.1 5374 kb 145 07641-L20557 GATA4 AAGAACCTGAAT-AAATCTAAGACA 8p23.1 6052 kb 454 19219-L25269 MTUS1 GCCTCGCTACTT-TCTGCAGCTTGT 8p22 2159 kb 154 11721-L21358 LPL TTTCCTCCCCAA-CAGTCTTCCATT 8p21.3 291 kb 312 02552-L25663 LZTS1 GCTGCAGCGCAA-GAAGAACGAGGC 8p21.3 2780 kb 481 19033-L24843 RHOBTB2 GTTGCATTTGTT-TTGTGGGCATTT 8p21.3 11 kb 241 17475-L21781 TNFRSF10B GGTGATTGTACA-CCCTGGAGTGAC 8p21.3 652 kb 353 17627-L25330 NKX3-1 CCTGTGTCACTG-AATATCAACTCT 8p21.2 1272 kb 217 14108-L25272 NEFL GGAACAAGCAGC-TAAGAAGAAAGA 8p21.2 1418 kb 139 18548-L25326 PPP2R2A AAGCCTCGCACA-GTTCTGAAGCCT 8p21.2 6421 kb 368 19028-L25331 NRG1 AAAACTTTCTGT-GTGAATGGAGGG 8p12 4970 kb 261 «17595-L25667 ZNF703 CACTTTGGGCCT-AAGCCGGTACCA 8p12 578 kb 319 19027-L25662 WHSC1L1 AAGATTACTGTT-TTCAATGTGGAG 8p12 181 kb 373 01046-L00624 FGFR1 CAACCTCTAACT-GCAGAACTGGGA 8p12 10595 kb 333 05757-L25660 PRKDC GGTGAAGTTCAT-CCTAGTGAGATG 8q11.21 8160 kb 246 01037-L00621 MOS CTGTGGCCATAA-AGCAAGTGAACA 8q12.1 4737 kb 279 06759-L25327 CHD7 AGGAACAGACAT-GCTAGCAGATGG 8q12.2 5716 kb 178 07915-L12249 MYBL1 CTGTTGACTGAA-GACATTTCAGAC 8q13.1 3489 kb 445 12002-L12830 PRDM14 CACTCTGGAGAC-AGACCATACCAG 8q13.3 108 kb 172 09940-L10399 NCOA2 GCGTGGCAAGAA-GAGTTCCCATGA 8q13.3 3793 kb 196 18147-L22651 TCEB1 ATGGCATCAGGT-CCTTCACAGCCA 8q21.11 6082 kb 202 01116-L00620 TPD52 CTTTGTTCTGCT-ACCCACTGCCAG 8q21.13 5194 kb 274 01039-L00617 E2F5 GCACCTTCTGGT-ACACAACTGGAG 8q21.2 4540 kb 135 17619-L25271 RIPK2 GCTTGGCCATTG-AGATTTCGCATC 8q21.3 4646 kb 472 19032-L24842 RAD54B GTCCAAGGAACA-AGAAGAGAAATC 8q22.1 3250 kb 292 04151-L23263 MTDH ACCTCAAAGTGT-AACAGCAAAGCA 8q22.1 4564 kb 338 11590-L15225 RRM2B GCTTTCAAATTC-TCATCGAGAATG 8q22.3 427 kb 166 «19026-L24836 KLF10 AGAAAAGTGATT-TTGAAGCTGTAG 8q22.3 6899 kb 400 19029-L24839 EBAG9 CCACCATGGCCA-TCACCCAGTTTC 8q23.2 7094 kb 432 01108-L25659 EIF3H TTCACTGCCCAA-AACTTAGGCAAG 8q24.11 1155 kb 346 03080-L02480 EXT1 TCCCAGTGCAGA-TCCACTCATCAG 8q24.11 6676 kb 284 04222-L16047 RNF139 ATCATCGACGCC-ATCTTCAACTCC 8q24.13 3265 kb 189 14869-L21679 MYC AACAACCGAAAA-TGCACCAGCCCC 8q24.21 0.1 kb 160 00580-L22108 MYC GAACGAGCTAAA-ACGGAGCTTTTT 8q24.21 2314 kb 299 16241-L18499 ASAP1 TTCCTTTCAGGC-TGTCCTTCGATG 8q24.21 2077 kb 391 06606-L06164 KCNQ3 AGACCATCCACA-TCAGAAATCGAA 8q24.22 906 kb 229 00488-L25274 SLA TCAGCTATGGCC-TTCGAGAGAGCA 8q24.22 189 kb 463 19031-L24841 WISP1 GATCTTGCCTAA-ATCCCAAGAAAT 8q24.22 31 kb 418 19030-L25658 NDRG1 CCAGGAAGCATT-TCAGCCAGCTGA 8q24.22 2290 kb 382 01047-L00622 KHDRBS3 TTCCATCTCAAT-GATGATCTCCAT 8q24.23 5152 kb 325 01042-L25661 PTK2 CTGGTTCACATG-GAATCACAGCCA 8q24.3 725 kb 254 17603-SP0531- AGAGGTTGCCTA-40nt spanning oligo- PTP3A4 L25332 GTGAGGCTGTGG 8q24.3 3203 kb 211 «01036-L00609 RECQL4 AGGCAAACGTTT-TCAAAACCTCGA 8q24.3 - SALSA probemix P014 Chromosome 8 Page 4 of 6
«This probe is located within, or close to, very strong CpG islands. A low signal of this probe can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. Note: Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. Table 2b. P014 reference probes arranged according to chromosomal location Length SALSA MLPA Partial sequence MV location Gene Location (nt) probe (24 nt adjacent to ligation site) (HG18) 266 04483-L03872 SLC2A1 1p34 GCCATCTTTTCT-GTTGGGGGCATG 01-043,169314 504 09870-L19465 PEX13 2p15 TGAGGATGACCA-TGTAGTTGCCAG 02-061,126361 184 06587-L06145 SCN2A 2q24 TGGTCAACAACT-ACAGTGAGTGCA 02-165,939588 130 16316-L18705 RAB7A 3q21 CACAATAGGAGC-TGACTTTCTGAC 03-129,999518 123 18709-L13645 IL4 5q31 ATCGACACCTAT-TAATGGGTCTCA 05-132,037613 409 10681-L11263 PKHD1 6p12 TCACAGCTGGTT-TCCTGAAAGGCT 06-051,932696 223 08064-L25273 DNAI1 9p13 AGGTGATTTCAG-AAACAGGAAACC 09-034,480008 361 06708-L25279 ZNF25 10p11 CAGGTGATTCCT-GGGGCTGCCAGC 10-038,300693 305 07022-L23200 RPGRIP1 14q11 GCTCAGGGCAGA-GCTGAAGGAAGA 14-020,849856 439 16538-L21570 SPG11 15q15 GTCAAAGGATTC-CCCGTTACTACT 15-042,671892 490 14909-L17529 RNMT 18p11 TACAATGAACTT-CAGGAAGTTGGT 18-013,724478 150 03797-L25270 KCNJ6 21q22 CTCGAAGCTCCT-ACATCACCAGTG 21-037,919598 Note: Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA MLPA Probemix P014-B1 Chromosome 8 sample picture Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analyzed with SALSA MLPA probemix P014-B1 Chromosome 8 (lot B1-0916). Implemented Changes compared to the previous product description versions. Version 10 23 June 2017 (T08) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2a/b, new picture included SALSA probemix P014 Chromosome 8 Page 5 of 6
- New references added on page 1 - Small changes of probe lengths in Tables 1 and 2a/b: in order to better reflect the true lengths of the amplification products. Version 09 03 March 2016 (T08) - Various minor textual and layout changes. - New reference added on page 2. Version 08 (52) - Product description adapted to a new product version (version number changed, lot number added, changes in Table 1 and Table 2, new picture included). - Various textual changes on page 1. - New reference was added on page 1. SALSA probemix P014 Chromosome 8 Page 6 of 6