Download SALSA MLPA KIT ME003-A1 Tumor suppressor-3 - MRC

MRC-Holland
Description version 04; 05-02-2010
®
MLPA
SALSA MLPA KIT ME003-A1 Tumor suppressor-3
Lot 0608
This SALSA® MLPA® kit is for basic research! This kit enables you to detect aberrant methylation of CpG
islands upstream of genes for which an altered methylation status in one or more types of tumours has been
reported in literature. In case interesting results are obtained by users, it is possible to develop methylation
kits specific for a certain tumour in collaboration with MRC-Holland. Interpretation of results obtained with
this product can be complicated. MRC-Holland cannot provide assistance with data interpretation.
Aberrant methylation of CpG-islands has been shown to be associated with transcriptional inactivation of
tumour suppressor genes in a wide spectrum of human cancers. CpG-islands are located in or near the
promoter region or other regulatory regions of approximately 50% of human genes.
This ME003-A1 MS-MLPA probemix contains 27 MS-MLPA probes which detect the methylation status of
promotor regions of 18 different tumour suppressor genes. These tumour suppressor genes are frequently
silenced by methylation in tumours, but are unmethylated in blood-derived DNA of healthy individuals. In
addition, 13 reference probes are included which are not affected by HhaI digestion. Besides detecting
aberrant methylation, all 40 probes present will give information on copy number changes in the analyzed
sample. The MLPA reaction requires as little as 20 ng of human DNA and can be used on a variety of DNA
samples, including those derived from paraffin-embedded tissues. Please note that each MS-MLPA reaction
generates two samples that need analysis by capillary electrophoresis: one undigested sample for copy
number detection and one digested sample for methylation detection. More information about MS-MLPA can
be found on page 2 and in the MS-MLPA protocol.
The MS-MLPA probes in this ME003-A1 probemix detect sequences in promoter regions of tumor suppressor
genes that are unmethylated in most blood-derived DNA samples. Upon digestion, the peak signal obtained
in unmethylated samples will be very small or absent. In contrast, when tested on in vitro methylated
human DNA, these probes do generate a signal. We have no data showing that methylation detected by a
particular probe indeed influences the corresponding mRNA levels.
This SALSA® MS-MLPA® kit can be used to detect aberrant methylation of one or more sequences of the
Tumour suppressor genes. Methylation levels can be different for different tissues. Please use DNA derived
from the same type of tissue and purified by the same method as the reference samples. This SALSA®
MLPA® kit can be used 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 3550% reduced relative peak area 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 area, 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. Finally,
note that most defects in this gene are expected to be small (point) mutations which will not be detected by
this SALSA MLPA test. We have no information on what percentage of defects in these genes is caused by
deletions/duplications of complete exons.
SALSA® MS-MLPA® kits are sold by MRC-Holland for research purposes and to demonstrate the
possibilities of the MLPA technique. This kit is not CE/FDA certified for use in diagnostic
procedures. SALSA MLPA kits are supplied with all necessary buffers and enzymes. Purchase of
the SALSA MLPA test kits includes a limited license to use these products for research purposes.
The use of this SALSA® MLPA® kit 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 Acid Research 30, e57 (2002). The MS-MLPA method for the detection of both copy numbers and
methylation changes was described in Nucleic Acid Research 33, e128 by Nygren et al. 2005.
More information
Website : www.mlpa.com
E-mail
: [email protected] (information & technical questions); [email protected] (for orders)
Mail
: MRC-Holland bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands
SALSA MLPA kit ME003 Tumor suppressor-3
Page 1 of 7
MRC-Holland
Description version 04; 05-02-2010
®
MLPA
Related SALSA MLPA kits
ME001 Tumor suppressor-1: Contains probes for Tumor suppressor genes.
ME002 Tumor suppressor-2: Can be used to confirm the results of ME001.
ME004 Tumor suppressor-4. Contains probe for Tumor suppressor genes.
Methylation-specific MLPA
Please note that each MS-MLPA reaction generates two samples that need analysis by capillary
electrophoresis: one undigested sample for copy number detection and one digested sample for methylation
detection.
A modification of the MLPA technique, MS-MLPA allows the detection of both copy number changes and
unusual methylation levels of 10-50 different sequences in one simple reaction. MLPA probes for methylation
quantification are similar to normal MLPA probes, except that the sequence detected by the MS-MLPA probe
contains the sequence recognized by the methylation-sensitive restriction enzyme HhaI. Similar to ordinary
MLPA reactions, the MS-MLPA protocol starts with sample DNA denaturation and overnight hybridization.
The reaction then is split into two tubes. One tube is processed as a standard MLPA reaction. This reaction
provides information on copy number changes. The other tube of the MLPA hybridization reaction is
incubated with the methylation-sensitive HhaI endonuclease while simultaneously, the hybridized probes are
ligated. Hybrids of (unmethylated) probe oligonucleotides and unmethylated sample DNA are digested by
the HhaI enzyme. Digested probes will not be exponentially amplified by PCR and hence will not generate a
signal when analyzed by capillary electrophoresis. In contrast, if the sample DNA is methylated, the hemimethylated probe-sample DNA hybrids are prevented from being digested by HhaI and the ligated probes
will generate a signal. More information about MS-MLPA can be found in the MS-MLPA protocol.
Please note that this product can not be used with an alternative protocol in which the genomic DNA is first
digested with Hha1, followed by MLPA reactions on both digested and undigested genomic DNA.
Data analysis
The ME003-A1 tumour suppressor probemix contains 40 MLPA probes with amplification products between
136 and 481 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.
The analysis of MS-MLPA kits consists of two parts: 1) determining copy numbers by comparing different
undigested samples (all MLPA kits), and 2) determining methylation patterns by comparing each undigested
sample to its digested counterpart (MS-MLPA kits only). The second part is unique for MS-MLPA kits and
serves to semi-quantify the percentage of methylation within a given sample.
Intra-sample data normalisation (all samples)
For analysis of MLPA results, not the absolute fluorescence values but “intra-normalized” data are used
(relative peak areas). The data generated in the digested and undigested sample should first be
normalized intra-sample by dividing the signal of each probe by the signal of every reference probe in
that sample, thus creating as many ratios per 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.
This Normalization Constant can then be used for sample to reference sample comparison, both for copy
number and digestion determination.
Copy numbers determination (comparison between undigested samples)
The final probe ratio, or ploidy status, of each probe is calculated by comparing a) the intra-normalized ratio
of each probe obtained on the undigested patient sample with b) the average intra-normalized ratio of each
probe obtained on the undigested reference samples.
Methylation analysis (comparing digested and undigested samples)
Methylation status of MS-MLPA probes* is calculated by dividing a) the Normalization Constant of each MSMLPA probe obtained on the digested patient sample by b) the Normalization Constant of each MS-MLPA
probe obtained on the corresponding undigested sample. Multiplying this value by 100 gives an estimation of
SALSA MLPA kit ME003 Tumor suppressor-3
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MRC-Holland
Description version 04; 05-02-2010
®
MLPA
the percentage methylation. Aberrant methylation can then be identified by comparing the methylation
status of one or more MS-MLPA probes in the sample in question to that obtained on reference samples.
*Note: An MS-MLPA probe targets a single specific HhaI site in a CpG island; if methylation is absent for a
particular CpG-site, this does not necessarily mean that the whole CpG island is unmethylated! For samples
containing both tumour and normal cells, MLPA experiments will indicate the average copy number of genes.
Data used for normalisation should have been obtained within a single experiment. Only samples from the
same tissue and purified by the same method should be compared. Confirmation of most exons deletions
and amplifications can be done by e.g. Southern blots, long range PCR, qPCR, FISH.
Note that Coffalyser, the MLPA analysis tool developed at MRC-Holland, can be downloaded free of charge
from our website www.mlpa.com.
This probemix was developed by A. Errami at MRC-Holland. In case the results obtained with this probemix
lead to a scientific publication, it would be very much appreciated if the probemix designer could be made a
coauthor. Info/remarks/suggestions for improvement: [email protected].
SALSA MLPA kit ME003 Tumor suppressor-3
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MRC-Holland
®
MLPA
Description version 04; 05-02-2010
Table 1. SALSA MS-MLPA ME003-A1 Tumour suppressor-3 probemix
Length (nt)
64-70-76-82
88-92-96
100
105
136
142
148
157 +
166
178
184
193
202
211
220
229
238
247
256
265
274
283
292
301
310
319
330
337
346 ٨
355
364
373
382
391
400
409
418
427
436
445
454
463
472
481
SALSA MLPA probe
Hha1 site
Chromosomal
position
Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA
D-fragments: Low signal of 88 or 96 nt fragment indicates incomplete denaturation
X-fragment: Specific for the X chromosome
Y-fragment: Specific for the Y chromosome
Reference (DNAH5) probe 08035-L07816
CCND2 probe 03312-L09381
SCGB3A1 probe 03305-L09382
BNIP3 probe 07138-L12958
DLC1 probe 02753-L02202
Reference (PTK2) probe 08236-L08114
DLC1 probe 02754-L02203
HLTF probe 02758-L02207
Reference (MLH3) probe 01245-L00793
SFRP5 probe 09149-L03207
CCND2 probe 03313-L02668
Reference (PAH) probe 02334-L01820
H2AFX probe 08511-L08607
CACNA1G probe 10123-L10466
Reference (BCL2) probe 00587-L00382
SFRP4 probe 03744-L03204
TWIST1 probe 02080-L02886
BCL2 probe 10352-L10890
Reference (NF1) probe 02524-L01955
CACNA1A probe 09055-L09224
Reference (CDK6) probe 03184-L02523
TIMP3 probe 10354-L10892
SFRP5 probe 09148-L12957
Reference (CDH1) probe 02416-L01862
ID4 probe 04497-L03909
H2AFX probe 08509-L08605
Reference (AI651963) probe 01234-L00781
RUNX3 probe 11131-L03905
SFRP4 probe 09147-L03205
Reference (KLK3) probe 00713-L00108
TIMP3 probe 10357-L10895
SCGB3A1 probe 11132-L12956
Reference (ATM) probe 02670-L02137
PRDM2 probe 09146-L02862
TGIF probe 02850-L13256
Reference (CD27) probe 00678-L00124
HLTF probe 09152-L09384
ID4 probe 04496-L03908
Reference (SLC6A5) probe 08946-L09041
RARB probe 10362-L10900
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
5p15.2
12p13.3
5q35
10q26.3
8p22
8q24
8p22
3q25.1
14q24.3
10q24.1
12p13.3
12q23
11q23.3
17q21.33
18q21.33
7p14.1
7p21.2
18q21.3
17q11.2
19p13.2
7q21.3
22q12.13
10q24.1
16q22.1
6p22.3
11q23.3
10p14
1p36.11
7p14.1
19q13.33
22q12.13
5q35
11q23
1p36.21
18p11.31
12p13.31
3q25.1
6p22.3
11p15.1
3p24.2
+ Results obtained with the BNIP3 probe should be disregarded as this probe probably also recognizes a
sequence on chromosome 14. Will be replaced in the next lot.
٨ This probe is not completely digested in DNA samples derived from blood.
Note: Please notify us of any mistakes. The identity of the genes detected by the reference probes is
available on request: [email protected].
SALSA MLPA kit ME003 Tumor suppressor-3
Page 4 of 7
MRC-Holland
®
MLPA
Description version 04; 05-02-2010
Table 2. ME003 probes arranged according to chromosomal location
Length
(nt)
SALSA MLPA
probe
Gene
HhaI
site
Chr & kb
to p-telo
373
11131-L03905
RUNX3
+
01-025.1
427
09146-L02862
PRDM2
+
01-013.8
481 »
10362-L10900
RARB
+
03-025.4
454
09152-L09384
HLTF
+
03-150.2
193
02758-L02207
HLTF
+
03-150.2
136
08035-L07816
DNAH5
148
03305-L09382
SCGB3A1
+
05-179.9
409
11132-L12956
SCGB3A1
+
05-179.9
346
04497-L03909
ID4
+
06-019.9
463
04496-L03908
ID4
+
06-019.9
265
03744-L03204
SFRP4
+
07-037.9
382
09147-L03205
SFRP4
+
07-037.9
274
02080-L02886
TWIST1
+
07-019.1
310
03184-L02523
CDK6
184
02754-L02203
DLC1
+
08-013.03
166
02753-L02202
DLC1
+
08-013.03
178
08236-L08114
PTK2
08-141.8
364
01234-L00781
AI651963
10-011.01
211
09149-L03207
SFRP5
+
10-099.5
330
09148-L12957
SFRP5
+
10-099.5
157 +
07138-L12958
BNIP3
+
10-133.6
472
08946-L09041
SLC6A5
11-020.6
418
02670-L02137
ATM
11-107.6
238
08511-L08607
H2AFX
+
11-118.4
355
08509-L08605
H2AFX
+
11-118.4
220
03313-L02668
CCND2
+
12-004.2
142
03312-L09381
CCND2
+
12-004.2
445
00678-L00124
CD27
12-006.4
229
02334-L01820
PAH
12-101.7
202
01245-L00793
MLH3
14-074.5
337
02416-L01862
CDH1
16-067.4
292
02524-L01955
NF1
17-026.6
247
10123-L10466
CACNA1G
05-013.9
07-092.08
+
SALSA MLPA kit ME003 Tumor suppressor-3
17-045.9
(partial) sequence with HhaI site
GTGCTTGGGTCTACGGGAATACGCATAACAGCGGCCGTCAGGGCGCCGGGCAGGCGGA
GCTGGGTGGTGGCCATTGGGCGACGGCGCAGGGTCAAGGGCCGGGCTCTGGGATCGTG
CATGTGCTTTTTCTGGAGTGGAAAAATACATAAGTTATAAGGAATTTAACAGACAGAAAGGCGCACAGAGGAATTT
CGGCTCGAAAACGATCCAGGGGAGCCGAGGCGCTCCTCTTGTCATCCCACTCAGCGCCAT
GACTGGACTCGCGGCGACTTACCTTTCAGTCGTGCGCTCCTGATCCGGCGCTCGGAATTTG
TTGTGGCTTCCTGTTTGGACCTGAACAAAACCGAAGTGGAGGATGCCATTCTTGAAGGGAATCA
GGGCCTCGAGGACTTCCTCTTGGCAGGCGCTGGGGCCCTCTGAGAGCAGGCAGGCCCGGCCTT
CCAGGAGGGCGGCGAGCTTCATGGCGCGGGGGCTCGGGGCGCGCGGGGAACCT
GGGAAATATAGAACAGAATGAACGTGCCTTCTTGAACAGCGCGTCTTTCTTAAGGCACTGGAATCCC
GTAGTGGAGGAGGCGCGGTTGTGAGTAGTACCGGGAGTGGGGTGATCCCGGGCTAGGGGAGCGC
GAAAGATGAGGGTGGCAGGAAGAGAAGGGCGCTTTCTGTCTGCCGGGGTCGCAGCGCGAGAGGG
CGGGATAAATAGGGTCCCGCAATGGCCGTGGCTGGCTGCGCTCCGAGCTGCGGAGTCC
CGCAGACTTCTTGCCGCGCTTGCCCTGGGCCGGGCTGCCCGGCTCGTCGCCG
GCGTGATTGGACTCCCAGGAGAAGAAGACTGGCCTAGAGATGTTGCCCTTCCCAGGCAGGCTTTTCA
GCCAATAGTTGGCGACTTTTCGGCGCTTCCCGCTGGGAACGTGGAGGCCCGTGGGGGAAAC
CAAGGATGCGTTGAGGACCGCGAGGGCGCGCGTCTCGGGTGCCGCCGTGGGT
TCTGACAGTGACGGCACCATCCCTAACCATTGCGGAGAATATGGCTGACCTAATAGATGGGTACTGCCGG
CAATTGCCATTTTTTCCTGACATTCACTGTGGAAATTTGGTGCACGACACTGTTAGGGGAGATCTGT
GTCCCGCCCAGCGCCAGGTCTTGCGCCCCTCTCCCGGCTCGGCTCCCCTGCCGCA
CGCTCGGCCTGCCAGCCATAGTAGTCGTACTCCTCGCAGCGCGCCGGCGCCCAGTGCAGCGC
GGCGCGGACTCCGATCGCCGCAGTTGCCCTCTGGCGCCATGTCGCAGAACGGAGC
CCATGACCTATGGCTCTTACCGCTATCCTAACTGGTCCATGGTGCTCGGATGGCTAATGCTCGCCTGTTC
GCATATCATAGACCTTGGTAGCAGTCTCTCTTTGCTGTGCCATCCCACTGTCTCTGGTACCCTCATAGGC
CCAACCGGAGGCGGGTATTGGAGAAAAGAGCCAATCAGGAGGGCGCAGAGGTGTGTCCTGGGGGCTT
GATTGTAGTGTTTAAATCTCGCGCGCTTTACAAGGATTGGCTACATCAGCGGATAGTTGGCAGTCTG
CAGGGAGAAAGCCTGGCGAGTGAGGCGCGAAACCGGAGGGGTCGGCGAGGATGCGGGCGAAGGA
CTGCATCGGTGTGGCCACGCTCAGCGCAGACACCTCGGGCGGCTTGTCAGCAGATGCAGG
GAAAGTCCTGTGGAGCCTGCAGAGCCTTGTCGTTACAGCTGCCCCAGGGAGG
CAGTGCCCTGGTTCCCAAGAACCATTCAAGAGCTGGACAGATTTGCCAATCAGATTCTCAG
GCGACCTTGTTCTTCCTTTCCTTCCGAGAGCTCGAGCAGAGAGGACTGTGATGAGACAGGATAACAG
CTATGAAGGAAGCGGTTCCGAAGCTGCTAGTCTGAGCTCCCTGAACTCCTCAGAGTCAGACAAAGACCAGGAC
GGCAATCCGGAATCCTCTGGAGTGGCACTGCAAGCAAATGGATCATTTTGTTGGACTCAATTTCAACT
CGGGCGATCCGGAGAGGGGCA-
Page 5 of 7
MRC-Holland
®
MLPA
Description version 04; 05-02-2010
Length
(nt)
SALSA MLPA
probe
Gene
HhaI
site
Chr & kb
to p-telo
436
02850-L13256
TGIF
+
18-003.4
283
10352-L10890
BCL2
+
18-059.1
256
00587-L00382
BCL2
301
09055-L09224
CACNA1A
391
00713-L00108
KLK3
400
10357-L10895
TIMP3
+
22-031.5
319
10354-L10892
TIMP3
+
22-031.5
(partial) sequence with HhaI site
AGCGGCGCCCCTCAGAGGAGGTGTCCTCACGCAA
CGTTTCTGTGGGAGGCCCTGAAACGCGCGGAGCTTCCCTCTGCCTCCAGGCTTTCCCAGCGAG
GTGAAGCGGTCCCGTGGATAGAGATTCATGCCTGTGCCCGCGCGTGTGTGCGCGCGTG
TTCTCCTGGCTGTCTCTGAAGACTCTGCTCAGTTTGGCCCTGGTGGGAGCTTGCATC
CGGGGCGCAAAGGATGTACAAGCAGTCAATGGCGCAGAGAGCGCGGACCATG
TGTGTCACCATGTGGGTCCCGGTTGTCTTCCTCACCCTGTCCGTGACGTGGA
CCAGCGCTATATCACTCGGCCGCCCAGGCAGCGGCGCAGAGCGGGCAGCAGGCAG
CATCGTGCTCCTGGGCAGCTGGAGCCTGGGGGACTGGGGCGCCGAGGCGTGCACA
18-058.9
+
19-013.4
19-056.05
+ Results obtained with the BNIP3 probe should be disregarded as this probe probably also recognizes a
sequence on chromosome 14. Will be replaced in the next lot.
» A second probe for this gene, recognizing a different CpG-site, is present in the SALSA MLPA kit ME002.
The Hha1 sites are marked with grey. Ligation sites are marked with –
Note: Complete probe sequences are available on request: [email protected]. Please notify us of any
mistakes: [email protected].
SALSA MLPA ME003-A1 Tumour suppressor-3 sample pictures
70000
301,23
60000
164,63
328,60
245,96
155,50
381,32
177,64
50000
193,40
237,51
85,53
40000
105,56
95,96
134,76
182,82
280,00
228,55
91,14
30000
398,80
218,25
209,31
148,66
140,78
354,76
291,35
201,13
452,74
256,39
273,43
264,15
20000
100,71
318,55
345,46
336,97
371,26
483,17
474,85
409,32
426,71
308,82
389,77
418,48
436,48
364,13
443,52
463,55
Dye Signal
10000
0
100
150
200
250
300
Size (nt)
350
400
450
Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng undigested human male
control DNA analyzed with SALSA MLPA kit ME003-A1 Tumor suppressor-3 (lot 0608).
SALSA MLPA kit ME003 Tumor suppressor-3
Page 6 of 7
MRC-Holland
®
MLPA
Description version 04; 05-02-2010
134.80
70000
177.46
60000
291.22
96.77
50000
201.14
91.52
228.52
256.24
40000
105.77
336.92
474.10
309.08
389.86
30000
100.97
418.44
86.08
443.72
364.17
20000
10000
106.73
Dye Signal
61.64
0
100
150
200
250
300
350
400
450
500
Size (nt)
Figure 2. Capillary electrophoresis pattern from a sample of approximately 50 ng digested human male
control DNA analyzed with SALSA MLPA kit ME003-A1 Tumor suppressor-3 (lot 0608).
Implemented Changes – the following has been altered compared to the previous product
description version(s).
Version 04 (05)
- ‘Basic research’ note added on page 1
- Columns in Table 2 have been adjusted. Textual changes on page 1 and 2, tables have been numbered.
SALSA MLPA kit ME003 Tumor suppressor-3
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