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MLPA Analysis FAQ How can I analyse my MLPA data? How is relative quantification on DNA kits performed? How is relative quantification on RNA kits performed? How is relative quantification on Methylation specific kits performed? I don't see any bands, what could be wrong? The peaks of the amplification products are visible, but very weak - how can I improve this? Why are there large differences in relative peak areas between different samples? How is it possible that I see extra peaks? Why do I see a blind peak? Why do the longer probe have smaller peak areas/lower peak heights? How come I find too many peaks with a length of >100 bp? My results don't make any sense.
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12. My results don't make any sense. Unfortunately, the MLPA technique is more sensitive to impurities than ordinary PCR. Results should therefore always be analysed by an experienced user and, if possible, be confirmed on a independently obtained DNA sample or by another technique. Most difficulties arise by comparing DNA samples purified by different methods or purified in different laboratories, since these often contain small amounts ionic impurities or very small remnants ethanol, phenol etc.. Although such impurities have little influence on ordinary PCR, they may result in small distortions of the MLPA multiplex PCR. Contaminants like proteins do not affect the MLPA reaction, as good results can be obtained directly on cell lysates. Most impurities, however, have their effect on the PCR reaction. Some of the ionic impurities can have an effect on DNA denaturation by increasing the Tm. For instance, the presence of 0.2 mM MgCl2 in a sample can prevent complete denaturation of strong CpG islands and will thereby especially affect the (usually GC-rich) exon 1 results of some genes. In case results seem unreliable, we recommend repeating the MLPA reaction starting from the very beginning. If no alternative DNA sample is available, it can be useful to perform a second MLPA reaction on two different amounts of the same DNA sample. Fortunately, if an MLPA reaction has gone wrong it is usually easily detected on the basis of its strange and unexpected results, such as the disappearances of many or all peaks in a sample, the warning signal of control peaks, or by the fact that an unusual high number of samples have the same abnormality. - back to top -
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1. How can I analyse my MLPA data? The first step is to generate raw data from your sequencer. Run & extraction protocols for the Beckman and ABI sequencers are available on our website: http://www.mrc-holland.com/pages/support_mlpa_analysispag.html. The second step entails the analysis of MLPA raw data. Analysis of a limited number of samples can be done by simple visual examination of the capillary electrophoresis peak profiles. A two colour on-screen overlay of sample and control reaction can be accomplished by Genotyper software, e.g. by changing the values for the length of the size markers by adding 2 nt to the control sample. However, for the analysis of large numbers of samples a more thorough analysis is usually desirable. Free software for the analysis of the great number of samples can be downloaded from the support website. In addition, information about other methods for analysis, including sheets made by users and commercially available software, can be found on the site as well. -back to top-
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2. How is relative quantification on DNA kits performed? At MRC-Holland, we export the peak sizes/peak areas obtained to an Excel file (freely downloadable from our website). During the first analysis step, non-specific amplification products, primer dimer peaks and the peaks generated by the MLPA control mix can usually be easily eliminated on the basis of their markedly lower peak area and/or small length. With only the peaks of the expected MLPA products left, the second step is then to normalise these remaining peak areas. Normalisation can be done by dividing the peak area of each amplification product by the combined peak area of all peaks in that sample, so that per sample, the ratio of a particular peak area of amplification product X to the whole is known. During the third step, these normalised peak areas are then compared to the average results obtained on a control sample, or (if this is possible) to the average results of all samples. The latter method can only be used if there aren't too many aberrant samples; see for counterexamples below. Furthermore, in order to obtain a valid comparison, results of samples with unreliable results should be removed from analysis. Divide the normalised peak area of a given probe amplification product X in sample A by the average normalised peak area of product X of a control, or by the average normalised peak area of product X of all samples. Finally, the results thus obtained can then be visualised using Excel graphics. Results differing by more than 20% are highlighted. Raw data of samples giving many aberrations should be checked; the following scenarios are possible. Very low peaks of both the MLPA products and the size standard may indicate a failure of injection in the capillary. Low MLPA products peaks in combination with high residual primer peaks may indicate a failure of the PCR reaction. High MLPA control peaks (64, 70, 76, 82 bp) but low MLPA product peaks indicate insufficient DNA sample or a failure of the ligation reaction. A deletion of one copy of a probe target sequence (autosomal chromosomes) will usually be apparent by a reduction in relative peak area for that probe amplification product of 35-55%. A gain in copy number from two to three copies or a diploid genome will usually be apparent by an increase in relative peak area between 30 and 55%. Standard deviation of peak areas should be below 10% for all probes. As said, analysis methods may differ for different applications. For example, in the case of our MSH2/MLH1 exon deletion probe mix P003, 19 out of the 42 probes detect the MLH1 gene. In case a MLH1 deletion spans one or a couple of exons, this will result in the reduction of the relative probe signal of the affected probe(s) by 40-55%. However, samples in which one copy of the complete MLH1 gene is deleted show a reduction of the relative probe signals of less than 40%. In this case, the MLH1 probe signals should be compared to the probe signals of all non-MLH1 probes, in order to obtain proper results. Similarly, the probe signals of the three ERBB2 (her2-neu) probes in our ERBB2 amplification assay P004 should be compared to the probe signals of the non-ERBB2 probes. -back to top-
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3. How is relative quantification on RNA kits performed? Analysis of mRNA MLPA results is different from the procedure on DNA samples. The expression level of different mRNA's usually varies. Therefore, large amounts of primers consumed by one or more abundant probe amplification products will result in a decrease in absolute signals obtained from other probes. However, by including probes for control mRNAs, assumed to be present in constant amounts, the amounts of certain mRNAs relative to these control mRNAs can be determined. For mRNA analysis, we assume one mRNA to be present in constant amounts. Relative probe signal of each probe is then defined by ratio of each measured peak area to the peak area of the reference probe. In our experience, beta-2-microglobulin (B2M) is a good reference gene for most studies. At MRC-Holland, we export the peak sizes /peak areas obtained to an Excel file (freely downloadable from our website). During the first analysis step, non-specific amplification products, primer dimer peaks and the peaks generated by the MLPA control mix can usually be easily eliminated on the basis of their markedly lower peak area and/or small length. With only the peaks of the expected MLPA products left, the second step is then to normalise by dividing each peak area by the peak area of the B2M control probe in that lane. Thirdly, these normalised peak areas are compared to the average results obtained on all samples. The normalised peak areas are divided by the average normalised peak area of a specific probe amplification product of all samples. All results differing by more than 30 % are highlighted. Raw data of samples that give many aberrations are checked. Furthermore, in order to obtain a valid comparison, results of samples with unreliable results should be removed from analysis. Again, for each sample, the normalised peak areas are divided by the average normalised peak area of that probe amplification product of all samples. Finally, the results thus obtained can then be visualised using Excel graphics. Raw data of samples giving many aberrations should be checked; the following scenarios are possible. Very low peaks of both the MLPA products and the size standard may indicate a failure of injection in the capillary. Low MLPA products peaks in combination with high residual primer peaks may indicate a failure of the PCR reaction. High MLPA control peaks (64, 70, 76, 82 bp) but low MLPA product peaks indicate insufficient sample or a failure of the ligation reaction. -back to top-
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4. How is relative quantification on Methylation specific kits performed? At MRC-Holland, we export the peak sizes / peak areas obtained to an Excel file (freely downloadable from our website). During the first analysis step, non-specific amplification products, primer dimer peaks and the peaks generated by the MLPA control mix can usually be easily eliminated on the basis of their markedly lower peak area and/or small length. With only the peaks of the expected MLPA products left, the second step is then to normalise these remaining peak areas. Normalisation for the analysis of copy numbers can be done by dividing the peak area of each amplification product by the combined peak area of all peaks of the control probes without Hha1 sites in that sample. So per sample, the ratio of a particular peak area of amplification product X to the control probes without Hha1 sites is known. During the third step, these normalised peak areas are then compared to the average results obtained on a control sample, or (if this is possible) to the average results of all samples. The latter method can only be used if the percentage of aberrant samples is low. Furthermore, in order to obtain a valid comparison, results of samples with unreliable results should be removed from analysis. Divide the normalised peak area of a given probe amplification product X in sample A by the average normalised peak area of product X of a control, or by the average normalised peak area of product X of all samples. Finally, the results thus obtained can then be visualised using Excel graphics. Results differing by more than 20% are highlighted. Quantification of the methylation status of a particular CpG site can be done by exporting the peak sizes and areas to an Excel file. Peak area of each MS-MLPA probe should be normalized by dividing it by the combined areas of the nearest control probes. Finally, the relative peak area of each target probe from the digested sample should be compared with those obtained from the undigested sample. Aberrant methylation can be identified as the appearance of a signal peak after HhaI digestion that otherwise was absent in normal, blood-derived, DNA. Raw data of samples giving many aberrations should be checked; the following scenarios are possible. Very low peaks of both the MLPA products and the size standard may indicate a failure of injection in the capillary. Low MLPA products peaks in combination with high residual primer peaks may indicate a failure of the PCR reaction. High MLPA control peaks (64, 70, 76, 82 bp) but low MLPA product peaks indicate insufficient DNA sample or a failure of the ligation reaction. A deletion of one copy of a probe target sequence (autosomal chromosomes) will usually be apparent by a reduction in relative peak area for that probe amplification product of 35-55%. A gain in copy number from two to three copies or a diploid genome will usually be apparent by an increase in relative peak area between 30 and 55%. Standard deviation of peak areas should be below 10% for all probes. -back to top-
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5. I don't see any bands, what could be wrong? 1. Is the molecular weight marker pattern ok? If not, check capillaries and electrophoresis conditions. 2. Is the PCR primer-peak present, and off-scale? If not, check whether the correct fluorescent label is used. Usually, FAM label is used for ABI apparatus, Cy5.0 for Beckman CEQ and IR800 for LICOR. 3. Are the four control fragments visible? Each SALSA probe mix will generate 4 fragments of 64, 70, 76 and 82 bp even when ligation is omitted (see next page). These bands will be small when more than 50 ng human DNA is used for MLPA. If the peak height of these bands is more than 50% of the peak height of the 92 bp (ligation-dependent) control band or the longer MLPA probe bands, either the ligation reaction has failed or the amount of sample DNA was much lower than the recommended minimum of 20 ng. In both cases, the results obtained may not be reliable. 4. Primer peak ok, but no control fragments and no probe amplification products: PCR reaction failed. We recommend repeating the PCR reaction using the same ligation reactions, but for instance using a different thermal cycler. Reduce all volumes by 50% to save reagents. 5. Primer peak ok, control fragments clearly visible, but no probe amplification products: 1. No DNA was present in your sample. 2. There could be a problem with the ligation reaction: although MLPA reagents are quite stable (after storage of a complete MLPA kit for 12 weeks at room temperature, good results were still obtained), it has been found that the Ligase cofactor NAD - present in Ligase buffer A - can occasionally be inactivated by a large number (<20) freeze-thaw cycles. Similarly, the polymerase and ligase might be inactivated by repeated freeze-thaw cycles when stored in freezers colder than -20 C (above this temperature, the enzymes remain liquid) and especially in freezers fluctuating between -25/ -30 C. -back to top-
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6. The peaks of the amplification products are visible, but very weak - how can I improve this? The tubes of the PCR reaction can be placed in the thermal cycler for another 4 cycles. Alternatively, performing a new PCR using the same ligation reactions but with 36-40 cycles might help. Reduce all volumes of the PCR reaction by 50% to save reagents. Increasing the number of PCR cycles to 40 will only have a very minor effect on the relative peak areas! -back to top-
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7. Why are there large differences in relative peak areas between different samples? 1. This might be due to impurities in the DNA preparations. The use of lower amounts of sample often solves the problem. 2. Another possible cause is overloading of capillaries, which can result in saturation of the fluorescence detection device. 3. Peak broadening due to deteriorated capillaries can influence results, especially when peak heights (instead of peak areas) are used for analysis. -back to top-
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8. How is it possible that I see extra peaks? 1. Incomplete denaturation during the electrophoresis can result in extra peaks or broader peaks, especially of longer fragments. An increase in the run temperature might solve this. 2. In some apparatus such as ABI3700, overloading of capillaries may result in signals in neighbouring capillaries. -back to top-
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9. Why do I see a blind peak? There are various probemixes which, when used on a no-DNA control sample, do not give a completely blank result. This is because of the presence of aspecific peaks. Usually, we release a probemix if the peak heights of any aspecific extra peaks in a DNA negative control are less than 50% of the four control peaks at 64-70-76-82 nt. Hence, when no DNA is used, these little extra peaks may still be visible. However, with increasing amounts of sample DNA, both the size of the small control peaks and of possible aspecific peaks will decrease. When 20 ng or more human DNA is used for analysis, the four control peaks at 64-70-76-82 nt will be small and any aspecific amplification products will have decreased peak areas that will not influence the results obtained. No DNA control sample - Ligation-independent background MLPA control reactions that completely lack sample DNA, or in which the Ligase-65 enzyme is omitted, should theoretically not yield any amplification products larger than 100 bp but are in practice often not completely blank. However, PCR is notorious to find always something to amplify. Compared to ordinary PCR reactions, MLPA is more prone to the generation of aspecific long amplification products in the absence of sample. Even without any ligation event, the M13 derived probe oligonucleotides are linearly amplified as the unlabeled PCR primer will bind to the long phosphorylated probe oligonucleotide and be elongated. In each PCR cycle, the complement of one of the M13 derived probe oligonucleotides and is elongated by the polymerase, this elongation product contains both PCR primer sequences and will be exponentially amplified in the next PCR cycles. This can result in an amplification product longer than 100 nucleotides (Ligation-independent background). This is the reason why at least 20 ng human DNA should be used in MLPA reactions and this is also the reason why the "no dna blank" is not always completely clean. We compare the intensity of aspecific amplification products in the no dna control reactions with the intensity of the small control fragments at 64-70-76 and 82 nt. When the aspecific amplification products that are longer than 120 nt are less than 50% in peak area as compared to the four small control fragments of 64, 70, 76 and 82 nt, new lots will be approved. Please note that the aspecific amplification products in a "no dna" control reaction will be almost absent in MLPA reactions on > 20 ng sample DNA. Their relative peak height will decrease in samples with increasing amounts of sample DNA, similar to that of the 64-70-76-82 nt control fragments which are almost invisible in samples containing > 50 ng sample DNA. No DNA control sample - Ligation-dependent background A control reaction in which the sample DNA is omitted may yield a few ligation-dependent amplification products larger than 100 bp. In a typical MLPA reaction, approximately 20.000 copies of the human haploid genome are present as well as 600.000.000 molecules of each probe and 25.000.000.000 molecules M13 single stranded DNA. In a few rare cases we observed a limited amount of probe ligation without template, probably due to ligation of probe oligonucleotides while annealed to other probe molecules or to the M13 DNA. If no specific ligation products are present, even extremely small amounts of inadvertently ligated probes will give amplification products when using a PCR reaction with 30 or more cycles. This ligation-dependent background is higher when ligation reactions are allowed to proceed for more than the recommended 15 minutes, or are left at temperatures lower than 50 °C. Contamination If there appears to be a large number of >100 bp amplification products present in your sample, one of your reagents might be contaminated by amplification products of previous MLPA reactions. Be sure never to use micro-pipettes that have been used for handling MLPA PCR products when setting up a new MLPA reaction! PCR reaction vials should never be opened in the vicinity of the thermal cycler or the lab area where the MLPA reactions are prepared. As with other PCR reactions, it is necessary to exercise extreme care to prevent contamination in your lab. -back to top-
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10. Why do the longer probe have smaller peak areas/lower peak heights? This can have several causes. 1. The volume of the DNA sample + probe mix +MLPA buffer is 8 µl. Due to the heated lid, evaporation during the 16 hr. hybridisation reaction is low. In general, more than 6-6.5 µl fluid remains at the bottom of the tube after this 16 hr incubation. However, if the volume decreases to less than 5 µl - e.g. due to bad closure of the tubes, incorrect heated lid temperature etc. - "fading" of the peak heights of the longer probes can occur. 2. Centrifugation of a mixture of MLPA buffer and probe mix can cause the larger probes to precipitate. Mix therefore by repeated pipetting. 3. Impurities in DNA samples most strongly affect longer probes and probes that already have a lower than average peak area. 4. The electrokinetic injection procedure of some capillary electrophoresis seems to select for injection of smaller fragments. This appears to be more pronounced with ABI sequencers as compared to the Beckman CEQ. 5. The use of older capillaries often results in a relatively low peak area of the longer probes. In some cases, this "ski slope" or fading effect can even be seen in the peak pattern of the molecular markers. -back to top-
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11. How come I find too many peaks with a length of >100 bp? If there appears to be a large number of >100 bp amplification products present in your sample, one of your reagents might be contaminated by amplification products of previous MLPA reactions. Be sure never to use micro-pipettes that have been used for handling MLPA PCR products when setting up a new MLPA reaction! A typical level of contamination that would yield amplification products might be less than 20 molecules. In a typical 50 µl MLPA PCR reaction, approximately 100.000.000.000 molecules of each probe will be generated. As with other PCR reactions, it is necessary to exercise extreme care to prevent contamination in your lab. -back to top-
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