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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 (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 microns</td>
			<td>Filmar</td>
			<td>74011</td>
			<td>http://www.filmar.qc.ca/index.php?filet=produits&#38;id=51&#38;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. 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’ interpretation and integration.

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

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

Flow-sorting and Exome Sequencing of the Reed-Sternberg Cells of Classical Hodgkin Lymphoma

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically comprise less than 1% of the tumor mass. Material derived from bulk tumor contains tumor content at a concentration insufficient for characterization. Therefore, fluorescence activated cell sorting using eight antibodies, as well as side- and forward-scatter, is described here as a method of rapidly separating and concentrating with high purity thousands of HRS cells from the tumor for subsequent study. At the same time, because standard protocols for exome sequencing typically require 100-1,000 ng of input DNA, which is often too high, even with flow sorting, we also provide an optimized, low-input library construction protocol capable of producing high-quality data from as little as 10 ng of input DNA. This combination is capable of producing next-generation libraries suitable for hybridization capture of whole-exome baits or more focused targeted panels, as desired. Exome sequencing of the HRS cells, when compared against healthy intratumor T or B cells, can identify somatic alterations, including mutations, insertions and deletions, and copy number alterations. These findings elucidate the molecular biology of HRS cells and may reveal avenues for targeted drug treatments.

Here, we describe a combined flow cytometric cell sorting and low-input, next-generation library construction protocol designed to produce high-quality, whole-exome data from the Hodgkin Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (CHL).

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically ...

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species.
Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family,&#160;it could be easily used to other prokaryote and eukaryote species

Here, we present a protocol for tracing genomic DNA (gDNA) contamination in RNA samples. The presented method utilizes primers specific for the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) genes. The method is suited for reliable and sensitive detection of DNA contamination in most eukaryotes and prokaryotes.

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but especially on red blood cells (RBCs). As a receptor for the complement components C3b and C4b, CR1 regulates the activation of the complement cascade and promotes the phagocytosis of immune complexes and cellular debris, as well as the amyloid-beta (A&#946;) peptide in Alzheimer's disease (AD). Several studies have confirmed AD-associated single nucleotide polymorphisms (SNPs), as well as a copy-number variation (CNV) in the CR1 gene. Here, we describe an innovative method for determining the length polymorphism of the CR1 receptor. The receptor includes three domains, called long homologous repeats (LHR)-LHR-A, LHR-C, and LHR-D-and an n domain, LHR-B, where n is an integer between 0 and 3. Using a single pair of specific primers, the genetic material is used to amplify a first fragment of the LHR-B domain (the variant amplicon B) and a second fragment of the LHR-C domain (the invariant amplicon). The variant amplicon B and the invariant amplicon display differences at five nucleotides outside of the hybridization areas of said primers. The numbers of variant amplicons B and of invariant amplicons is deduced using a quantitative tool (high-resolution melting (HRM) curves), and the ratio of the variant amplicon B to the invariant amplicon differs according to the CR1 length polymorphism. This method provides several advantages over the canonical phenotype method, as it does not require fresh material and is cheaper, faster, and therefore applicable to larger populations. Thus, the use of this method should be helpful to better understand the role of CR1 isoforms in the pathogenesis of diseases such as AD.

Here, we describe an innovative method to determine complement receptor 1 (CR1) length polymorphisms for use in several applications, particularly the assessment of susceptibility to diseases such as Alzheimer&#39;s disease (AD). This method could be useful to better understand the role of CR1 isoforms in the pathogenesis of AD.

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...