The authors thank Sophie Patrier and Marianne Par&#233;sy for sharing the original flow cytometry protocol, and Promega and Qiagen for providing supplies and reagents. This work was supported by the R&#233;seau Qu&#233;b&#233;cois en Reproduction and the Canadian Institute for Health Research (MOP-130364) to R.S.

<ol>
	<li>Szulman, A. E., Surti, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+syndromes+of+hydatidiform+mole.+II.+Morphologic+evolution+of+the+complete+and+partial+mole.">The syndromes of hydatidiform mole. II. Morphologic evolution of the complete and partial mole.</a> American Journal of Obstetrics &amp; Gynecology. 132 (1), 20-27 (1978).</li><li>Fukunaga, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interobserver+and+intraobserver+variability+in+the+diagnosis+of+hydatidiform+mole.">Interobserver and intraobserver variability in the diagnosis of hydatidiform mole.</a> The American Journal of Surgical Pathology. 29 (7), 942-947 (2005).</li><li>Gupta, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+reproducibility+of+hydatidiform+moles:+ancillary+techniques+(p57+immunohistochemistry+and+molecular+genotyping)+improve+morphologic+diagnosis+for+both+recently+trained+and+experienced+gynecologic+pathologists.">Diagnostic reproducibility of hydatidiform moles: ancillary techniques (p57 immunohistochemistry and molecular genotyping) improve morphologic diagnosis for both recently trained and experienced gynecologic pathologists.</a> The American Journal of Surgical Pathology. 36 (12), 1747-1760 (2012).</li><li>Howat, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+histopathologists+reliably+diagnose+molar+pregnancy?.">Can histopathologists reliably diagnose molar pregnancy?.</a> Journal of Clinical Pathology. 46 (7), 599-602 (1993).</li><li>Banet, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+hydatidiform+moles:+analysis+of+a+prospective+series+with+p57+immunohistochemistry+and+molecular+genotyping.">Characteristics of hydatidiform moles: analysis of a prospective series with p57 immunohistochemistry and molecular genotyping.</a> Modern Pathology. 27 (2), 238-254 (2014).</li><li>Lipata, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+DNA+genotyping+diagnosis+of+hydatidiform+mole.">Precise DNA genotyping diagnosis of hydatidiform mole.</a> Obstetrics &amp; Gynecology. 115 (4), 784-794 (2010).</li><li>Buza, N., Hui, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+hydatidiform+mole:+histologic+parameters+in+correlation+with+DNA+genotyping.">Partial hydatidiform mole: histologic parameters in correlation with DNA genotyping.</a> International Journal of Gynecologic Pathology. 32 (3), 307-315 (2013).</li><li>Fisher, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+of+heterozygous+complete+hydatidiform+moles,+estimated+by+locus-specific+minisatellite+and+Y+chromosome-specific+probes.">Frequency of heterozygous complete hydatidiform moles, estimated by locus-specific minisatellite and Y chromosome-specific probes.</a> Human Genetics. 82 (3), 259-263 (1989).</li><li>Murdoch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+NALP7+cause+recurrent+hydatidiform+moles+and+reproductive+wastage+in+humans.">Mutations in NALP7 cause recurrent hydatidiform moles and reproductive wastage in humans.</a> Nature Genetics. 38 (3), 300-302 (2006).</li><li>Parry, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+causing+familial+biparental+hydatidiform+mole+implicate+c6orf221+as+a+possible+regulator+of+genomic+imprinting+in+the+human+oocyte.">Mutations causing familial biparental hydatidiform mole implicate c6orf221 as a possible regulator of genomic imprinting in the human oocyte.</a> American Journal of Human Genetics. 89 (3), 451-458 (2011).</li><li>Nguyen, N. M., Slim, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+Epigenetics+of+Recurrent+Hydatidiform+Moles:+Basic+Science+and+Genetic+Counselling.">Genetics and Epigenetics of Recurrent Hydatidiform Moles: Basic Science and Genetic Counselling.</a> Current Obstetrics and Gynecology Reports. 3, 55-64 (2014).</li><li>Sebire, N. J., Savage, P. M., Seckl, M. J., Fisher, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathological+features+of+biparental+complete+hydatidiform+moles+in+women+with+NLRP7+mutations.">Histopathological features of biparental complete hydatidiform moles in women with NLRP7 mutations.</a> Placenta. 34 (1), 50-56 (2013).</li><li>Nguyen, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+genotype-phenotype+correlations+between+NLRP7+mutations+and+the+balance+between+embryonic+tissue+differentiation+and+trophoblastic+proliferation.">Comprehensive genotype-phenotype correlations between NLRP7 mutations and the balance between embryonic tissue differentiation and trophoblastic proliferation.</a> Journal of Medical Genetics. 51 (9), 623-634 (2014).</li><li>Brown, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+pregnancy+loss+in+a+woman+with+NLRP7+mutation:+not+all+molar+pregnancies+can+be+easily+classified+as+either+"partial"+or+"complete"+hydatidiform+moles.">Recurrent pregnancy loss in a woman with NLRP7 mutation: not all molar pregnancies can be easily classified as either "partial" or "complete" hydatidiform moles.</a> International Journal of Gynecologic Pathology. 32 (4), 399-405 (2013).</li><li>Colgan, T. J., Chang, M. C., Nanji, S., Kolomietz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Reappraisal+of+the+Incidence+of+Placental+Hydatidiform+Mole+Using+Selective+Molecular+Genotyping.">A Reappraisal of the Incidence of Placental Hydatidiform Mole Using Selective Molecular Genotyping.</a> The International Journal of Gynecological Cancer. 26 (7), 1345-1350 (2016).</li><li>Murphy, K. M., McConnell, T. G., Hafez, M. J., Vang, R., Ronnett, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genotyping+of+hydatidiform+moles:+analytic+validation+of+a+multiplex+short+tandem+repeat+assay.">Molecular genotyping of hydatidiform moles: analytic validation of a multiplex short tandem repeat assay.</a> The Journal of Molecular Diagnostics. 11 (6), 598-605 (2009).</li></ol>

The authors have nothing to disclose.

HM are abnormal human pregnancies with heterogeneous etiologies and have different histological and genotypic types, which makes their accurate classification and diagnosis challenging. Histopathological morphological evaluation was often proven inaccurate and is therefore unreliable on its own to classify HM into CHM and PHM and distinguish them from non-molar miscarriages. Therefore, an accurate diagnosis of HM requires the use of other methods such as multiplex microsatellite DNA genotyping, ploidy analysis by flow cytometry, ploidy analysis by FISH, and p57KIP2 immunohistochemistry. Each of these methods has its own limitations and advantages.
Limitations and advantages of multiplex genotyping and flow cytometry
Maternal contamination is one of the most common issues when working with FFPE tissues and may lead to misdiagnosis, thus highlighting the importance of separating maternal from POC tissues. It is important to identify maternal contamination in order to have an idea of what to expect of the genotyping peaks and to help with their interpretation. If the level of contamination is too large and prevents reliable interpretation of the results, then repeat the DNA isolation and extraction, taking extra care in removing all possible maternal tissues. In regions where the morphology of the tissues is not clear, it is better to remove such regions to minimize the chance of maternal contamination. The first advantage of the method described in this protocol, in addition to its lower cost, is that maternal tissues are removed from the slides while other methods consist of collecting POC tissues (by covering them with solution that polymerizes when exposed to air and lifting the tissues) without removing maternal tissues. The method described here thus allows one to take a second look at the remaining POC tissues, re-clean them if necessary, as is often the case, and then collect and extract DNA from them. The second advantage is that we clean tissues on uncoverslipped H&#38;E stained sections, which greatly facilitates the removal of maternal tissues, as opposed to the use of unstained sections and a coverslipped map slide. However, the additional staining may further degrade the DNA, and this is compensated for by adding more sections.
The availability of parental DNA for the analysis is another challenge. The presence of both parents greatly facilitates the analysis and interpretation of genotyping results. Unfortunately, however, it is quite often the case that the father&#39;s DNA is not available, which may sometimes complicate the analysis, especially in cases where the quality of the POC DNA is poor due to prior fixation or long-term storage (more frequent when working with recurrent HM). Furthermore, maternal blood may not always be available for DNA extraction. In such cases, maternal DNA can be extracted from maternal endometrial tissues present in the FFPE blocks as previously described16. Also, characterizing molar tissues that resulted from the use of assisted reproductive technologies may complicate microsatellite genotype analysis because, in most of the cases, DNA from the donors (males or females) is usually not available.
Lastly, the fact that larger peaks tend to be shorter in terms of peak height is another possible source of confusion, particularly when the peak heights need to be used to determine if there are two doses or one dose in a single peak. One way to overcome this is to always keep in mind that peaks of large allele size (number of base pairs) tend to be shorter, due to the degraded nature of DNA from FFPE tissues, which makes less DNA available for the amplification of larger alleles. In addition, a shorter PCR fragment amplification takes less time than a larger one, and the exponential amplification of DNA leads to fewer amounts of larger alleles.
The main drawback of flow cytometry is that triploid conceptions may sometimes be missed, and this could be due to insufficient amounts of CV in the block. However, the presence of a triploid peak is a conclusive indication of a triploidy. Note that this method is not sensitive enough to detect trisomies, tetraploid conceptions, or other aneuploidies using this protocol. Tetraploid conceptions are not detectable by this protocol because the tetraploid peak corresponds to the same peak of diploid cells in the G2 phase of the cell cycle.
Knowing these limitations and challenges helps in reducing mistakes. It is thus important and sometimes necessary to analyze the same tissue with different methods, compare the results, and make sure that they are concordant with one another. If they are not, results need to be reconsidered and analyses need to be repeated. For the several cases that showed conflicting results, discrepancies were resolved simply by repeating the experiments and taking appropriate care to avoid the original problem. In other cases, discrepancies were resolved by performing additional methods such as FISH on tissue sections or by performing additional simplex genotyping with appropriate markers.
Identification of maternal contamination
With respect to identifying contamination of the POC DNA with maternal DNA, the level of contamination will be reflected or recognized by the presence of all maternal alleles at all loci in the POC, in addition to the maternal allele(s) that are transmitted to the POC.
In Figure 11A, peaks originating from maternal DNA contamination are labelled with a &#34;c&#34;. Notice how every peak marked with a &#34;c&#34; is also present in the mother, and we see these &#34;c&#34; peaks in all three markers. Note that for the second marker (in blue), the maternal contamination peak indicated by a &#34;c&#34; is higher than each &#34;c&#34; peak at the first marker because the mother is homozygous for the second marker; the height of the contaminant is therefore doubled for this marker. In this POC, there are no maternally derived peaks. In other words, this POC did not inherit any alleles from the mother. There is only one real peak at each marker and this peak is not present in the mother. We therefore know that the real peaks must have come from the father. With ploidy information from either karyotype or flow cytometry analysis demonstrating diploidy, it is possible to conclude that this POC is both androgenetic in origin and diploid.
Furthermore, these three markers in Figure 11A reveal that there is always only one real peak for every marker. Only three markers are illustrated here, however, multiplex kits often come with many more markers that will also reveal the same pattern. Since this POC is diploid, there must be two doses in each peak. With this information, we can conclude that this POC is androgenetic monospermic and is homozygous at every single marker.
Figure 11B represents a diploid biparental POC. The little black bar across the first peak of the first marker (in green) represents the estimated amount of contamination that is present within this real peak. The &#34;R&#34; indicates the real portion of this peak coming from the POC DNA; the &#34;c&#34; represents the contaminant portion of this peak coming from the maternal DNA. How can one identify the level of contamination? It is possible, in this case, when a marker is heterozygous in the mother because one of the maternal alleles is absent in the POC. The small peak in the first marker labelled with &#34;c,&#34; for example, indicates that this is the level of contamination that should be expected for the other maternally inherited peak. Consequently, all the POC peaks that are of maternal origin are slightly higher than the paternally inherited peaks due to the small added amount of contamination. For the third marker (in black) in Figure 11B, the level of contamination is expected to be double the usual amount (because the mother is homozygous for this specific marker), and one can therefore infer that there is only one dose in the first peak in the POC.
Figure 11C illustrates a triploid dispermic POC. The first marker (in green) shows three peaks in the POC. Since these three peaks have similar heights, it is possible to conclude that this POC is likely triploid. Note that the third peak in this marker is shorter than the first. This is expected because, as a general trend, larger peaks tend to be shorter. The second peak is slightly higher than the first, and this can be accounted for by a small amount of maternal contamination, as indicated by the bar and &#34;c.&#34; Furthermore, two of these three peaks are not present in the mother, and therefore must have come from the father. Thus far, this marker indicates that the POC could be triploid (or a trisomy) and that the origin of the extra set of chromosomes is paternal. It also indicates that it is a dispermic conception, since the POC inherited two different alleles from the father.
The second marker (in blue) in Figure 11C has only two peaks. After accounting for the level of contamination, the second peak appears higher than the first, and this is despite the general trend for larger peaks to be smaller (a trend that must always be kept in mind during analysis). Thus, it is likely that there are two doses in that one big peak. This is also supported by the fact that the first marker is indicative of triploidy.
Lastly, the third marker (in black) in Figure 11C shows three peaks, again indicative of triploidy. Since most markers in multiplex kits come from different chromosomes, it is possible to conclude with confidence that a POC is triploid after observing the same trend of three alleles across several different markers. Also note from this marker that two out of the three peaks originate from the father, confirming the dispermic origin.
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/60366/60366fig11a.jpg" /> 
Figure 11: Genotyping results illustrating the effect of maternal contamination. The top panels show the alleles that belong to the POC and the bottom panels show those that belong to the mother. In (A), the POC is androgenetic monospermic and the level of contamination is highlighted by an arrow and the letter &#34;c.&#34; In (B), the POC is diploid biparental, and the level of contamination is highlighted by the letter &#34;c.&#34; In (C), the POC is triploid dispermic and the level of contamination is highlighted by the letter &#34;c.&#34; The little black bars show how much of the peak heights comes from maternal contamination and should therefore be taken into consideration when comparing peak heights. The x-axis is in basepairs; the labels and basepair sizes have been omitted for simplicity. The y-axis represents peak height and is similarly omitted from the figure for simplicity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60366/60366fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Examples of challenging cases
For a number of cases, it was only possible to arrive at a conclusion by having all three assessments simultaneously (i.e., flow, p57KIP2, and multiplex genotyping). For example, one case (808) was referred as a non-molar miscarriage. Histopathological evaluation lead to suspicion of a molar conception and genotyping analysis of the POC revealed one marker that clearly showed three peaks (two from the father and one from the mother). The p57KIP2 results were not conclusive. The flow cytometry analysis revealed a small triploid peak that was confirmed with FISH analysis, which also ruled out the presence of another diploid cellular population. With confirmation from FISH, it was possible to conclude that the POC is indeed a triploid dispermic mole.
Another case (1192) was also referred as non-molar miscarriage. The CV of this POC were intermingled with maternal tissues and were highly necrotic as well, making the cleaning process very challenging. Consequently, the first genotyping analysis showed a high level of maternal contamination, such that every maternal allele could be seen in the POC (a good indication of possible contamination). Furthermore, the p57KIP2 was unfortunately inconclusive, most likely due to the necrotic nature of the tissue, perhaps because of delayed fixation. The flow cytometry results, however, were indicative of triploidy, which was also confirmed by FISH. The DNA was re-extracted with the goal of reducing the level of contamination and removing tissues with unclear morphology. Analysis of the new genotyping results, while accounting for the probable presence of maternal contamination, allowed us to conclude that the POC was a triploid dispermic XXY PHM.
Table 4 outlines the typical methods used for analysis. It is recommended to use morphological evaluation and p57KIP2 immunohistochemistry on all tissues suspicious of HM and at least one genotyping method. Among genotyping methods, the most informative is multiplex DNA genotyping. When results between different methods are not concordant or when some results are inconclusive, other genotyping methods need to be used. The table demonstrates expected concordant results for each possible HM type along with rare exceptions and recommendations to solve them.
<img alt="Table 4" src="/files/ftp_upload/60366/60366table4a.jpg" />

Table 4: Typical order of analysis, expected results, and rare exceptions along with recommendations to solve them. P57KIP2 immunohistochemistry aims to detect the expression of p57KIP2, the protein coded by CDKN1C gene. This gene is paternally imprinted in the cytotrophoblast and the villous stroma of first trimester placenta and is expressed only from the maternal genome. It is therefore used as an ancillary marker to detect, in an easy and inexpensive way, the presence of the maternal genome in the POC. It is recommended to perform p57KIP2 immunohistochemistry for all POCs suspected to be HM in parallel to the morphological evaluation. In the author&#39;s laboratory, the p57KIP2 antibody and platforms indicated in the Table of Materials are used and the authors are very happy with the quality of the results. Abbreviations: IHC = immunohistochemistry; Dip And = diploid androgenetic; Dip Bip = diploid biparental; Chr = chromosome; MC = miscarriage; and TP = trophoblastic proliferation.
To the best of the authors&#39; knowledge, this article is the first to provide detailed protocols for flow cytometry as well as low-cost and high-quality multiplex microsatellite DNA genotyping of FFPE POC tissues. The interpretation of the results is also described, along with their troubleshooting and integration with those of other methods to reach accurate conclusions and diagnoses of POCs and HM. The authors sincerely hope that this article can be helpful for researchers trying to understand this complex entity.

A hydatidiform mole (HM) is an abnormal human pregnancy characterized by abnormal embryonic development, hyperproliferation of the trophoblast, and hydropic degeneration of chorionic villi (CV). Historically, HM used to be divided into two types, complete HM (CHM) and partial HM (PHM) based only on morphological evaluation1. However, it has been shown that morphological evaluation alone is not sufficient to classify HM into the two subtypes (CHM and PHM) and distinguish them from non-molar miscarriages2,3,4.
Because CHM and PHM have different propensities to malignancies, it is therefore important to accurately determine the genotypic type of HM to provide appropriate follow-up and management to the patients. Consequently, in the past decades, several methodologies have been developed and evolved for the purpose of identifying the parental contribution to the molar tissues and reaching a correct classification of HM. These include karyotype analysis, chromosomal banding polymorphism, human leukocyte antigen (HLA) serological typing, restriction fragment length polymorphism, variable number of tandem repeats, microsatellite genotyping, flow cytometry, and p57KIP2 immunohistochemistry. This has allowed accurate subdivision of HM conceptions based on the parental contribution to their genomes, as follows: CHM, which are diploid androgenetic monospermic or diploid androgenetic dispermic, and PHM, which are triploid, dispermic in 99% and monospermic in 1% of the cases5,6,7,8. Furthermore, there is another genotypic type of HM that emerged in the past two decades, which is diploid biparental. The latter is mostly recurrent and may affect a single family member (simplex cases) or at least two family members (familial cases). These diploid biparental moles are mostly caused by recessive mutations in NLRP7 or KHDC3L in the patients9,10,11,12. Diploid biparental HM in patients with recessive mutations in NLRP7 may be diagnosed as CHM or PHM by morphological analysis and this appears to be associated with the severity of the mutations in the patients13,14. In addition to the classification of HM according to their genotypes, the introduction and use of several genotyping methods allowed the distinction of the various molar entities from non-molar miscarriages, such as aneuploid diploid biparental conceptions and other types of conceptions5,15. Such conceptions may have some trophoblast proliferation and abnormal villous morphology that mimic, to some extent, some morphological features of HM.
The purpose of this article is to provide detailed protocols for multiplex genotyping and flow cytometry of formalin-fixed paraffin-embedded (FFPE) tissues, and comprehensive analyses of the results of these methods and their integration with other methods for correct and conclusive diagnosis of molar tissues.

<table><tbody><tr><td>BD FACS Canto II</td><td>BD BioSciences</td><td>338960</td><td></td></tr><tr><td>Capillary electrophoresis instrument: Genomes Applied Biosystems 3730xl DNA Analyzer</td><td>Applied biosystems</td><td>313001R</td><td>Service offered by the Centre for Applied Genomics&lt;br/&gt;(http://www.tcag.ca)</td></tr><tr><td>Citric acid</td><td>Sigma</td><td>251275</td><td></td></tr><tr><td>Cytoseal 60, histopathology mounting medium</td><td>Fisher</td><td>23244257</td><td></td></tr><tr><td>Eosin Y stock solution (1%)</td><td>Fisher</td><td>SE23-500D</td><td></td></tr><tr><td>FCSalyzer - flow cytometry analysis software</td><td>SourceForge</td><td>-</td><td>https://sourceforge.net/projects/fcsalyzer/</td></tr><tr><td>FFPE Qiagen kit</td><td>Qiagen</td><td>80234</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11295-51</td><td>For sectioning and for the cleaning process</td></tr><tr><td>Glacial Acetic Acid (Concentrated)</td><td>Sigma</td><td>A6283-500mL</td><td></td></tr><tr><td>Glass coverslips: Cover Glass</td><td>Fisher</td><td>12-541a</td><td></td></tr><tr><td>Hematoxylin</td><td>Fisher</td><td>CS401-1D</td><td></td></tr><tr><td>Highly deionized formamide: Hi-Di Formamide</td><td>Thermofisher</td><td>4311320</td><td></td></tr><tr><td>IHC platform: Benchmark Ultra</td><td>Roche</td><td>-</td><td></td></tr><tr><td>Kimwipes</td><td>Ultident</td><td>30-34120</td><td></td></tr><tr><td>Microtome</td><td>Leica</td><td>RM2135</td><td></td></tr><tr><td>Microtome blades</td><td>Fisher</td><td>12-634-1C</td><td></td></tr><tr><td>Nitex filtering mesh, 48 &amp;mu;m</td><td>Filmar</td><td>74011</td><td>http://www.filmar.qc.ca/index.php?filet=produits&amp;amp;id=51&amp;amp;lang=en ; any other filter is suitable, but this is an inexpensive and effective option from a non-research company</td></tr><tr><td>p57 antibody</td><td>Cell Marque</td><td>457M</td><td></td></tr><tr><td>Pasteur pipette</td><td>VWR</td><td>53499-632</td><td></td></tr><tr><td>PCR machine</td><td>Perkin Elmer, Applied Biosystems</td><td>GeneAmp PCR System 9700</td><td></td></tr><tr><td>PeakScanner 1.0</td><td>Applied Biosystems</td><td>4381867</td><td>Software for genotyping analysis.</td></tr><tr><td>Pepsin from porcine gastric mucosa</td><td>Sigma</td><td>P7012</td><td></td></tr><tr><td>Polystyrene round-bottom tubes</td><td>BD Falcon</td><td>352058</td><td></td></tr><tr><td>Positively charged slides: Superfrost Plus 25x75mm</td><td>Fisher</td><td>1255015</td><td></td></tr><tr><td>PowerPlex 16 HS System</td><td>Promega Corporation</td><td>DC2102</td><td></td></tr><tr><td>Propidium Iodide</td><td>Sigma</td><td>P4864</td><td></td></tr><tr><td>Ribonuclease A from bovine pancreas</td><td>Sigma</td><td>R4875</td><td></td></tr><tr><td>Separation matrix: POP-7 Polymer</td><td>Thermofisher</td><td>4352759</td><td></td></tr><tr><td>UltraPure Agarose</td><td>Fisher</td><td>16500-500</td><td></td></tr><tr><td>Xylene</td><td>Fisher</td><td>X3P1GAL</td><td></td></tr></tbody></table>

microsatellite dna genotyping and flow cytometry ploidy analyses of formalin-fixed paraffin-embedded hydatidiform molar tissues

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by excessive trophoblastic proliferation and abnormal embryonic development. There are two types of HM based on microscopic morphological evaluation, complete HM (CHM) and partial HM (PHM). These can be further subdivided based on the parental contribution to the molar genomes. Such characterization of HM, by morphology and genotype analyses, is crucial for patient management and for the fundamental understanding of this intriguing pathology. It is well documented that morphological analysis of HM is subject to wide interobserver variability and is not sufficient on its own to accurately classify HM into CHM and PHM and distinguish them from hydropic non-molar abortions. Genotyping analysis is mostly performed on DNA and tissues from formalin-fixed paraffin-embedded (FFPE) products of conception, which have less than optimal quality and may consequently lead to wrong conclusions. In this article, detailed protocols for multiplex genotyping and flow cytometry analyses of FFPE molar tissues are provided, along with the interpretation of the results of these methods, their troubleshooting, and integration with the morphological evaluation, p57KIP2 immunohistochemistry, and fluorescence in situ hybridization (FISH) to reach a correct and robust diagnosis. Here, the authors share the methods and lessons learned in the past 10 years from the analysis of approximately 400 products of conception.

The complexity of molar tissues and the fact that they may have various genotypes necessitates stringent analysis and the use of several methods such as morphological evaluation, p57 immunohistochemistry, microsatellite genotyping, flow cytometry, and FISH. For example, one patient (1790) was referred with two PHM that were found to be triploid by microarray analysis of the POCs only. The patient was therefore diagnosed with recurrent PHM. Microsatellite genotyping of her two &#34;PHM&#34; along with the DNA of the patient and her partner revealed that while the patient&#39;s first mole is triploid dispermic (Figure 9A), her second &#34;PHM&#34; has a triploid digynic genotype (Figure 9B) and is therefore not a PHM, but a non-molar miscarriage.
The first marker in Figure 9A (in black) shows two peaks in the POC. The first peak originates from the mother, since only the mother has a peak of this size. Following the same reasoning, the second peak originates from the father since he shares the same allele. Notice how the second peak is a lot higher than the first, indicating that there are probably two doses of the same paternal allele in that peak. Maternal contamination, which will be explained in more detail later on, is very minimal in this POC because the POC displays a very tiny peak at the position of the second maternal allele.
The second marker in Figure 9A (in blue) shows three peaks in the POC. Two of these peaks originate from the father and one from the mother. Thus, it is apparent again from this marker that there are three alleles present in the POC, two from the father and one from the mother. The third and fourth markers in Figure 9A are similar to the first marker, and also show two alleles coming from the father and one from the mother.
Since all four markers consistently show three alleles (by dose or by the presence of three alleles of different sizes), two originating from the father and one from the mother, one can conclude that this POC is triploid dispermic, and confirms the diagnosis of PHM.
All four markers in Figure 9B again show three alleles: the first marker shows two doses in the first peak that originate from the mother and one dose in the second peak that originates from the father. The second and fourth markers show three different peaks (i.e., three different alleles), two of which are from the mother. The third marker shows one dose in the first peak originating from the father and two doses in the second peak originating from the mother. Therefore, this POC is triploid digynic in origin since two sets of chromosomes come from the mother and one set comes from the father. It is therefore a non-molar miscarriage.
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/60366/60366fig9a.jpg" /> 
Figure 9: Representative genotyping results of patient 1790. (A) Select genotyping results of the patient&#39;s first conception showing a triploid dispermic genotype. (B) Select genotyping results from the patient&#39;s second conception showing a triploid digynic genotype. The x-axis is in basepairs; the labels and basepair sizes have been omitted for simplicity. The y-axis represents peak height and is similarly omitted from the figure for simplicity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60366/60366fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This patient was initially misdiagnosed with two PHM and was worrying about an increased risk of more moles while she had a single PHM. This case highlights the limitation of SNP microarray on the POC alone. SNP microarray is a powerful method and is the best to detect aneuploidies of any chromosome (trisomies, monosomies, or non-diploid genotypes); however, when performed on the POC alone without analyzing parental DNA, the origin of the triploidy cannot be determined. For further explanations about genotype analysis and interpretation, please refer to the study by Murphy et al.16.
Representative results shown in Figure 10A are of a triploid conception. The values of the x-axis represent the nuclear DNA content. For example, 200 is an arbitrary number given to cells that contain a certain amount of nuclear DNA content. Therefore, a peak at 400 represents cells that contain double the amount of nuclear DNA content as compared to the 200 peak. The little peak around 300 represents nuclear DNA content that is in between the 200 and 400 peak and is therefore the triploid peak. Notice how a diploid conception (Figure 10B) does not contain any peak at the 300 value.
In some cases, the triploid peak is very subtle (Figure 10C). Whenever a barely noticeable triploid peak is noted, it is important to first consider the amount of CV that were present in the sections used for the flow cytometry analysis. If the sections had less than about 20% CV, then it will likely be a true triploidy since it is expected to be a very low peak. If the sections taken had high amounts of CV, the POC becomes suspicious of mosaicism with the presence of another diploid cellular population. This can be checked by re-reviewing the genotyping results to see if they fit a perfect triploid dispermy or can also be confirmed by FISH with probes from the X, Y, and 18 chromosomes. Also, using the software described in this article, it is possible to set specific gates, as described in section 2.4.1, that allow the user to focus on a specific region to enrich for triploid cells if they really exist.
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/60366/60366fig10a.jpg" /> 
Figure 10: Representative flow cytometry results demonstrating triploid conceptions in (A) and (C), and a diploid conception in (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60366/60366fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Microsatellite DNA Genotyping and Flow Cytometry Ploidy Analyses of Formalin-fixed Paraffin-embedded Hydatidiform Molar Tissues at JoVE.com

Microsatellite DNA Genotyping and Flow Cytometry Ploidy Analyses of Formalin-fixed Paraffin-embedded Hydatidiform Molar Tissues

Hydatidiform moles are abnormal human pregnancies with heterogeneous aetiologies that can be classified according to their morphological features and parental contribution to the molar genomes. Here, protocols of multiplex microsatellite DNA genotyping and flow cytometry of formalin-fixed paraffin-embedded molar tissues are described in detail, together with results&#8217; interpretation and integration.

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by excessive trophoblastic proliferation and abnormal embryonic development. There are ...

microsatellite-dna-genotyping-flow-cytometry-ploidy-analyses-formalin

Nanyang Technological University

Research

JoVE Journal

Genetics

9.1K Views.  McGill University. Het belangrijkste voordeel van ons genotyperingprotocol is dat het besmetting met moeder-DNA minimaliseert, wat de belangrijkste uitdaging is bij het genotyperen van formeel vaste, paraffine-ingebedde molaire weefsels. Flow cytometrie is een snelle en goedkope methode die sterk vergemakkelijkt de diagnose van hydatidiform moedervlekken. Voor elke formaline-vaste paraffine-embedded, of FFPE, product van de conceptie, bereiden een vier-micron dikke hematoxylin en eosine vlek sectie zoals beschr...

Video: Microsatellite DNA Genotyping and Flow Cytometry Ploidy Analyses of Formalin-fixed Paraffin-embedded Hydatidiform Molar Tissues

FISH for Pre-implantation Genetic Diagnosis 

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

FISH for Pre-implantation Genetic Diagnosis

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

Medicine

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Developmental Biology

Transmitochondrial Cybrid Generation From Suspension-Growing Cancer Cells

Transmitochondrial Cybrid Generation Using Cancer Cell Lines

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more efforts are still needed to fully understand the link involving alterations in mitochondria and tumorigenesis, as well as to identify tumor-associated mitochondrial phenotypes. For instance, to evaluate the contribution of mitochondria in tumorigenesis and metastasis processes, it is essential to understand the influence of mitochondria from tumor cells in different nuclear environments. For this purpose, one possible approach consists of transferring mitochondria into a different nuclear background to obtain the so-called cybrid cells. In the traditional cybridization techniques, a cell line lacking mtDNA (&#961;0, nuclear donor cell) is repopulated with mitochondria derived from either enucleated cells or platelets. However, the enucleation process requires good cell adhesion to the culture plate, a feature that is partially or completely lost in many cases in invasive cells. In addition, another difficulty found in the traditional methods is achieving complete removal of the endogenous mtDNA from the mitochondrial-recipient cell line to obtain pure nuclear and mitochondrial DNA backgrounds, avoiding the presence of two different mtDNA species in the generated cybrid. In this work, we present a mitochondrial exchange protocol applied to suspension-growing cancer cells based on the repopulation of rhodamine 6G-pretreated cells with isolated mitochondria. This methodology allows us to overcome the limitations of the traditional approaches, and thus can be used as a tool to expand the comprehension of the mitochondrial role in cancer progression and metastasis.

Author Spotlight: Transmitochondrial Cybrid Generation Using Cancer Cell Lines  

This protocol describes a technique for cybrid generation from suspension-growing cancer cells as a tool to study the role of mitochondria in the tumorigenic process.

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more ...

Cancer Research

Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, along with a thermoplastic elastomer (TPE) microfluidic droplet generation device. Epigenetic markers are used for WBC&#160;subtyping which is of important prognostic value in different diseases. This is achieved through the quantification of DNA methylation patterns of specific CG-rich regions in the genome (CpG loci). In this paper, bisulfite-treated DNA from peripheral blood mononuclear cells (PBMCs) is encapsulated in droplets with mdPCR reagents including primers and hydrolysis fluorescent probes specific for CpG loci that correlate with WBC sub-populations. The multiplex approach allows for the interrogation of many CpG loci without the need for separate mdPCR reactions, enabling more accurate parametric determination of WBC sub-populations using epigenetic analysis of methylation sites. This precise quantification can be extended to different applications and highlights the benefits for clinical diagnosis and subsequent prognosis.

Epigenetic markers are used for white blood cell (WBC) subtyping through the quantification of DNA methylation patterns. This protocol presents a multiplex droplet polymerase chain reaction (mdPCR) method using a thermoplastic elastomer (TPE)-based microfluidic device for droplet generation allowing for precise and multiplex methylation-specific target quantification of WBC differential counts.

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, ...

Bioengineering

Single Oocyte Bisulfite Mutagenesis

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

Biology

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...

Flow Cytometric Analysis of Biomarkers for Detecting Human Sperm Functional Defects

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no abnormalities in conventional semen parameters. Additional technologies are needed to explain the idiopathic infertility and detect subtle sperm defects. Currently, biomarkers of sperm function, including sperm apoptosis, mitochondrial membrane potential (MMP), and DNA damage, reveal sperm physiology at the molecular level and are capable of predicting male fertility.
With flow cytometry (FCM) techniques, each of these markers can be rapidly, accurately, and precisely measured in human semen samples, but time costs substantially increase and results could be obstructed if all the biomarkers need to be tested with a single cytometer. In this protocol, after collection and immediate incubation at 37 &#176;C for liquefication, semen samples were further analyzed for sperm apoptosis using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The MMP was labeled with 5,5&#8242;,6,6&#8242;-tetrachloro-1,1&#8242;,3,3&#8242;-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) probe, and DNA damage was assessed using the sperm chromatin structure assay (SCSA) with acridine orange (AO) staining. Thus, flow cytometric analysis of sperm function markers can be a practical and reliable toolkit for the diagnosis of infertility and evaluation of sperm function at both bench and bed.

The present protocol provides a practical solution that allows for the measurement of apoptosis, mitochondrial membrane potential, and DNA damage in human sperm with a single cytometer.

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no ...

Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron transport chain. Unlike the diploid nuclear genome, most cells contain many more copies of mtDNA, ranging from less than 100 to over 200,000 copies depending on cell type. MtDNA copy number is increasingly used as a biomarker for a number of age-related degenerative conditions and diseases, and thus, accurate measurement of the mtDNA copy number is becoming a key tool in both research and diagnostic settings. Mutations in the mtDNA, often occurring as single nucleotide polymorphisms (SNPs) or deletions, can either exist in all copies of the mtDNA within the cell (termed homoplasmy) or as a mixture of mutated and WT mtDNA copies (termed heteroplasmy). Heteroplasmic mtDNA mutations are a major cause of clinical mitochondrial pathology, either in rare diseases or in a growing number of common late-onset diseases such as Parkinson's disease. Determining the level of heteroplasmy present in cells is a critical step in the diagnosis of rare mitochondrial diseases and in research aimed at understanding common late-onset disorders where mitochondria may play a role. MtDNA copy number and heteroplasmy have traditionally been measured by quantitative (q)PCR-based assays or deep sequencing. However, the recent introduction of ddPCR technology has provided an alternative method for measuring both parameters. It offers several advantages over existing methods, including the ability to measure absolute mtDNA copy number and sufficient sensitivity to make accurate measurements from single cells even at low copy numbers. Presented here is a detailed protocol describing the measurement of mtDNA copy number in single cells using ddPCR, referred to as droplet generation PCR henceforth, with the option for simultaneous measurement of heteroplasmy in cells with mtDNA deletions. The possibility of expanding this method to measure heteroplasmy in cells with mtDNA SNPs is also discussed.

Here we present a protocol for measuring absolute mitochondrial (mt)DNA copy number and mtDNA deletion heteroplasmy levels in single cells.

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron ...

Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms has increased in recent years due to the recently developed ability to produce models by mtDNA mutagenesis and a new appreciation of the association between mitochondrial genetic aberrations and common age-related diseases such as cancer, diabetes, and dementia. Pyrosequencing is a sequencing-by-synthesis technique that is widely employed across the mitochondrial field for routine genotyping experiments. Its relative affordability when compared to massive parallel sequencing methods and ease of implementation make it an invaluable technique in the field of mitochondrial genetics, allowing for the rapid quantification of heteroplasmy with increased flexibility. Despite the practicality of this method, its implementation as a means of mtDNA genotyping requires the observation of certain guidelines, specifically to avoid certain biases of biological or technical origin. This protocol outlines the necessary steps and precautions in designing and implementing pyrosequencing assays for use in the context of heteroplasmy measurement.

Pyrosequencing assays enable the robust and rapid genotyping of mitochondrial DNA single nucleotide polymorphisms in heteroplasmic cells or tissues.

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms ...

Microsatellite DNA Genotyping and Flow Cytometry Ploidy Analyses of Formalin-fixed Paraffin-embedded Hydatidiform Molar Tissues

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video