We acknowledge Amy Thomasson for her assistance with preparing this manuscript, and Fredrik Skarstedt and Ryan Christy for their efforts in the preparation of figures.

With the approval of the Institutional Review Board, a search of the electronic laboratory database was performed to identify formalin-fixed, paraffin embedded (FFPE) blocks representing cases of cytomegalovirus (CMV) infection in gastrointestinal (GI) biopsies diagnosed by histopathology (hematoxylin and eosin (H&#38;E) and/or immunohistochemistry (IHC)).
1. Tissue Processing from FFPE GI Biopsy Tissue
<ol>
	<li>Collect FFPE GI biopsy tissue blocks for cytomegalovirus (CMV) quantitative polymerase chain reaction (qPCR). These blocks are stored at room temperature.</li>
	<li>Pre-label 1.7 ml microfuge tubes with the block&#8217;s unique identification number.</li>
	<li>Lock the microtome handwheel.</li>
	<li>Seat the tissue block into the cassette clamp.</li>
	<li>Using a fresh, unused blade, align the block to the knife angle on the microtome.</li>
	<li>Adjust the microtome to cut 10 &#956;m&#160;thick sections.</li>
	<li>Unlock the handwheel and advance the block toward the blade.</li>
	<li>Cut 5 sections of the block at 10 &#956;m thick intervals, allowing each cut to roll into a scroll of tissue. Ensure that each scroll contains an adequate representation of the desired biopsy tissues.</li>
	<li>Lock the microtome handwheel.</li>
	<li>With forceps, place all 5 scrolls of tissue into the pre-labeled centrifuge tube, being careful to only manipulate the scroll by the paraffin in order to reduce potential for cross contamination. Snap the cap on the tube and place it back on the rack.</li>
	<li>Remove the tissue block from the microtome.</li>
	<li>Remove and discard the blade.</li>
	<li>Decontaminate any surfaces or instruments that may have come in contact with the previous tissue.</li>
	<li>Place a fresh, unused blade into the knife holder.</li>
	<li>Repeat steps 1.4-1.14 for each additional block to be processed. The microfuge tubes containing the tissue scrolls may be stored at room temperature until the time of DNA extraction.</li>
</ol>
2. DNA Extraction from Scrolls of FFPE GI Biopsy Tissue
NOTE: This protocol is adapted from the package insert of the FFPE tissue DNA extraction kit(CAUTION: refer to Material Safety Data Sheet (MSDS)).
<ol>
	<li>Add 1 ml xylene (CAUTION: Refer to MSDS) to each sample microfuge tube. Close the lid and vortex vigorously for 10 sec.</li>
	<li>Proceed with the DNA extraction as specifically outlined in the manufacturer&#8217;s product insert with the following modifications.</li>
	<li>First add 6 &#956;l of the internal control (IC) to each microfuge tube.
	<ol>
		<li>Following the ATL/proteinase K lysis incubation at 56 &#176;C for 1 hr, incubate at 99 &#176;C for 30 min, rather than 90 &#176;C for 1 hr.</li>
		<li>During the final DNA elution step, carefully open the lid of the DNA purification column and apply 50 &#956;l distilled DNAse/RNAse-free water at the center of the membrane, rather than buffer ATE. 
		NOTE: Refer to the package insert for the appropriate storage and preparation of reagents. DNA should be extracted from all 5 FFPE scrolls. Perform all centrifugation steps at room temperature (15-25 &#176;C).</li>
	</ol>
	</li>
</ol>
3. qPCR for CMV
<ol>
	<li>Polymerase Chain Reaction (PCR) Procedure
	<ol>
		<li>Generate an Excel PCR worksheet for the CMV assay run. This Excel worksheet calculates the total amount of each reagent needed to make up the master mix with Mg. It multiplies the amount of each reagent needed per sample (listed below) times the number of samples and controls plus one (to compensate for loss due to pipetting).
		<ol>
			<li>Include the following reagents in each PCR reaction in the amount indicated: 
			Reagent&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Amount per Sample 
			CMV LC master mix vial &#160; &#160; &#160; &#160; &#160; &#160; &#160; 12.5 &#956;l 
			Mg Solution yellow vial &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; 2.5 &#956;l 
			Total volume &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;15.0 &#956;l</li>
		</ol>
		</li>
		<li>Include in every assay, run a water or no target control (NTC), negative control, a low positive control, a high positive control, and either QS3 or QS4, and all patient samples. The capillaries should be set up in this order.</li>
		<li>Obtain the cooling block specific for the real-time PCR instrument that is stored at 4 &#176;C. Place the appropriate capillary tubes in the cooling block. Do not touch the surface of the capillary tubes. Always use gloves when handling the capillaries.</li>
		<li>Set up all polymerase chain reactions in a clean hood not contaminated with amplification products.
		<ol>
			<li>Remove the correct number of master vials from the PCR kit to test all patient samples, plus 5 controls. Let the master vials, Mg solution, and PCR grade water thaw in the cooling block. Each master vial contains enough reagents for 12 tests.</li>
			<li>Gently vortex the master vials and quick spin them in the centrifuge at the maximum speed.</li>
			<li>Combine the contents of each master vial into one vial, and gently vortex again.</li>
			<li>Combine the master mix and Mg solution in the amounts indicated on the PCR worksheet into a single sterile microfuge tube.</li>
			<li>Mix gently and aliquot 15 &#956;l of master mix with Mg into each capillary tube, except the capillary in position 5 which is for the quantitation standard (QS).</li>
		</ol>
		</li>
		<li>Move the cooling block containing the capillary tubes and master mix with Mg from the clean hood to a processing hood.</li>
		<li>Add 2 &#956;l of internal control (IC) to the remaining 30 &#956;l of master mix with Mg. Mix gently and aliquot 15 &#956;l into the capillary in position 5 (position for the QS).</li>
		<li>Pipette 10 &#956;l of water, control DNA, or sample DNA into the appropriate capillary tube. Cap the capillary tubes using the capping tool.</li>
		<li>Take cooling block/samples to the real-time PCR instrument.</li>
	</ol>
	</li>
	<li>Program the real-time PCR Instrument (see Table 1)</li>
	<li>Amplification and Detection performed on the real-time PCR instrument
	<ol>
		<li>Log onto the computer</li>
		<li>Double click on the instrument icon and log into the software.</li>
		<li>Turn on the real-time PCR instrument</li>
		<li>Click on &#8220;CMV/EBV&#8221; from the front screen.</li>
		<li>Follow the prompts of the macro wizard.</li>
		<li>Select the box to run a self-test when prompted by the software. A self-test must be performed once each day the instrument is in use.</li>
		<li>Name the run.</li>
		<li>Adjust the sample count located in the upper left corner of the page to the sum of controls and patients included in the run and enter the identification number of each patient sample.</li>
		<li>Click on the &#8220;Abs Quant&#8221; tab.</li>
		<li>Under &#8220;Sample type&#8221;, assign the appropriate sample type for the standard.</li>
		<li>Type in the expected concentrations for the quantitation standard (QS).</li>
		<li>Remove the capillary tubes from the cooler block and place them into the corresponding position in the carousel (cooling block sample #1 should be placed in position #1 on the carousel, etc.).</li>
		<li>Place the carousel into the instrument&#8217;s centrifuge and press start.</li>
		<li>Open centrifuge and remove the carousel.</li>
		<li>Place the carousel into the real-time PCR instrument. Close the lid.</li>
		<li>Press &#8220;Start Run&#8221;.</li>
		<li>The real-time PCR instrument will check each position for a sample. Do not walk away from the instrument until this check has been completed, especially if one of the capillaries is not in the carousel. The instrument will note this and ask if the run is to be continued. Answer yes before leaving.</li>
		<li>Once the run is complete, close the report that was generated.</li>
		<li>Click on &#8220;Absolute Quantitation&#8221;.</li>
		<li>Click on the arrow next to Color Compensation.</li>
		<li>Click &#8220;Select Color Compensation&#8221; from the drop-down box.</li>
		<li>Choose the most current color compensation file to apply to the run.</li>
		<li>Click OK when the box pops up.</li>
		<li>Click on the arrow by Standard Curve.</li>
		<li>Select &#8220;use external&#8221; from the drop-down menu.</li>
		<li>Select the most current external standard curve file, then click OK.</li>
		<li>Look at each curve under the Absolute Quantification menu to ensure that curves are present and not flat line, if a concentration is assigned. The Absolute Quantification for the 705/Back 530 is automatically analyzed.</li>
	</ol>
	</li>
	<li>Quality control
	<ol>
		<li>Generate a new standard curve whenever a new lot number of reagents is received, or every six months, whichever is more frequent.</li>
		<li>Add 10 &#956;l of each standard to the master mix with Mg. Consider the standards included with the kit as previously extracted DNA.</li>
		<li>Add 1 &#956;l of internal control for each standard to the master mix with Mg used in generating the standard curve in order to assure accurate quantitation. Do not add IC to the master mix with Mg for the No Template Control (NTC).</li>
		<li>Prepare a NTC and three replicates of Quantitation Standards QS 4, 3, 2, 1, and a 1:10 dilution of QS4 for the run to generate the standard curve, totaling in 16&#160;data points.</li>
		<li>Select &#8220;replicate of&#8221; in the drop down menu for repeated standards</li>
		<li>Choose channel 530/back none and turn on color compensation when analyzing the run,</li>
		<li>Under &#8220;Standard Curve (In Run)&#8221;, select &#8220;save as external&#8221; and name the curve. Enter the comment &#8220;applied as standard curve&#8221; when saving.</li>
		<li>Graph the results. The resulting linearity coefficient must be &#8805;0.81.</li>
		<li>For daily runs, include one copy of QS3 or QS4 as a control in the run. The current external standard curve may be imported into the run during the analysis phase.</li>
		<li>Run Controls: no DNA control, negative control, low, and high positive controls. Expected viral load is determined and adjusted for each vendor and lot number.</li>
		<li>Add an IC to each sample and control during the DNA extraction.</li>
		<li>If any of the controls fall out of range, record the unacceptable results and refer the assay for troubleshooting.</li>
	</ol>
	</li>
	<li>Frequency of controls
	<ol>
		<li>Include in every run: a NTC, negative, low positive, high positive, and standard control.</li>
	</ol>
	</li>
</ol>
NOTE: Acceptable limits for controls. No peaks should be observed for the no template (NTC) and negative controls. Peaks should be present in the 530 channel and expected viral loads are determined and adjusted for each vendor and lot number for the low and high positive controls.

<ol>
	<li>Hodinka, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of Clnical Microbiology. 2, 1558-1574 (2011).</li><li>Lasry, 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=Interstrain+variations+in+the+cytomegalovirus+(CMV)+glycoprotein+B+gene+sequence+among+CMV-infected+children+attending+six+day+care+centers.">Interstrain variations in the cytomegalovirus (CMV) glycoprotein B gene sequence among CMV-infected children attending six day care centers.</a> The Journal of infectious diseases. 174, 606-609 (1996).</li><li>Murph, J. R., Baron, J. C., Brown, C. K., Ebelhack, C. L., Bale Jr, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+occupational+risk+of+cytomegalovirus+infection+among+day-care+providers.">The occupational risk of cytomegalovirus infection among day-care providers.</a> JAMA : the journal of the American Medical Association. 265, 603-608 (1991).</li><li>Pass, R. 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=Vaccine+prevention+of+maternal+cytomegalovirus+infection.">Vaccine prevention of maternal cytomegalovirus infection.</a> The New England journal of medicine. 360, 1191-1199 (2009).</li><li>McCoy, M. H., 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=qPCR+increases+sensitivity+to+detect+cytomegalovirus+in+paraffin-embedded,+formalin-fixed+tissue+of+gastrointestinal+biopsies.">qPCR increases sensitivity to detect cytomegalovirus in paraffin-embedded, formalin-fixed tissue of gastrointestinal biopsies.</a> Human Pathology. 45, 48-53 (2014).</li><li>Mills, A. M., Guo, F. P., Copland, A. P., Pai, R. K., Pinsky, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+CMV+detection+in+gastrointestinal+mucosal+biopsies+using+immunohistochemistry+and+PCR+performed+on+formalin-fixed,+paraffin-embedded+tissue.">A comparison of CMV detection in gastrointestinal mucosal biopsies using immunohistochemistry and PCR performed on formalin-fixed, paraffin-embedded tissue.</a> The American journal of surgical pathology. 37, 995-1000 (2013).</li><li>Krech, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement-fixing+antibodies+against+cytomegalovirus+in+different+parts+of+the+world.">Complement-fixing antibodies against cytomegalovirus in different parts of the world.</a> Bulletin of the World Health Organization. 49, 103-106 (1973).</li><li>Chou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newer+methods+for+diagnosis+of+cytomegalovirus+infection.">Newer methods for diagnosis of cytomegalovirus infection.</a> Reviews of infectious diseases. 12 Suppl 7, (1990).</li><li>Brytting, M., Xu, W., Wahren, B., Sundqvist, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytomegalovirus+DNA+detection+in+sera+from+patients+with+active+cytomegalovirus+infections.">Cytomegalovirus DNA detection in sera from patients with active cytomegalovirus infections.</a> Journal of clinical microbiology. 30, 1937-1941 (1992).</li><li>Revello, M. G., 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=Human+cytomegalovirus+in+blood+of+immunocompetent+persons+during+primary+infection:+prognostic+implications+for+pregnancy.">Human cytomegalovirus in blood of immunocompetent persons during primary infection: prognostic implications for pregnancy.</a> The Journal of infectious diseases. 177, 1170-1175 (1998).</li><li>Shinkai, M., Bozzette, S. A., Powderly, W., Frame, P., Spector, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+urine+and+leukocyte+cultures+and+plasma+DNA+polymerase+chain+reaction+for+identification+of+AIDS+patients+at+risk+for+developing+human+cytomegalovirus+disease.">Utility of urine and leukocyte cultures and plasma DNA polymerase chain reaction for identification of AIDS patients at risk for developing human cytomegalovirus disease.</a> The Journal of infectious diseases. 175, 302-308 (1997).</li></ol>

The authors have nothing to disclose.

Many laboratory methods are available for the diagnosis of cytomegalovirus (CMV) infection, including serology, viral culture, molecular viremia assays, histology of biopsy material, and, as recently demonstrated, molecular assays of formalin-fixed, paraffin-embedded (FFPE) biopsy tissue5,6. Serology, once a mainstay of diagnosis, only reliably identifies exposure and does not correlate well with acute CMV infection7. Viral culture, both conventional and early antigen shell vial, is hampered by leng...

Interpretation of cytomegalovirus (CMV) infection in clinical specimens is critically important. CMV is a member of the betaherpesvirinae subfamily of herpesviruses. Similar to other herpesviruses, CMV has the ability to establish persistent or latent infection characterized by a lack of active viral replication1. Reactivation of virus may occur during times of stress or other immunosuppression, leading to active viral replication characterized by virion release, which may be accompanied by disease manifestations2-4. Gastrointestinal (GI) CMV disease symptoms frequently include abdominal pain and/or bloody stools1.
The diagnosis of CMV gastroenteritis by histology utilizes hematoxylin and eosin (H&#38;E) to identify CMV viral inclusions. In cases where clinical suspicion is high yet no obvious inclusions are observed, immunohistochemistry (IHC) is frequently used as an adjunct test method. However, IHC can also be hampered by rare non-classic appearing cellular staining patterns (equivocal), making interpretation difficult (Figure 2)5. It was sought to use DNA extracted from formalin-fixed, paraffin-embedded (FFPE) GI biopsy tissue and quantitative polymerase chain reaction (qPCR) to detect CMV in GI biopsies. This technique has been shown to be valuable in the detection of CMV in GI biopsies, and is particularly helpful in equivocal IHC staining cases5,6. Also, qPCR has also been shown to correlate well with additional clinical CMV test data when it is available6. Use of qPCR in detecting CMV may lead to earlier diagnosis and treatment in cases where clinical suspicion for CMV is high, but H&#38;E and CMV IHC are negative.

<table><tbody><tr><td>1.7 ml Microfuge tubes</td><td>Costar</td><td>3620</td><td></td></tr><tr><td>Serrated forceps</td><td>Electron Microscopy Services</td><td>62086-1S</td><td>Micro Forceps MF-1</td></tr><tr><td>Tabletop centrifuges (microfuge)</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Xylene</td><td>Fisher</td><td>Fisher Scientific: X3P</td><td>1 gallon</td></tr><tr><td>Ethanol</td><td>Fisher</td><td>Fisher Scientific: ET108</td><td>96-100%</td></tr><tr><td>Microtome</td><td>Leica</td><td>RM2255</td><td></td></tr><tr><td>QIAamp DNA FFPE Tissue Kit</td><td>Qiagen</td><td>56404</td><td></td></tr><tr><td>artus CMV LightCycler PCR ASR</td><td>Qiagen</td><td>4500025</td><td></td></tr><tr><td>CMV LC PCR Supplement Kit</td><td>Qiagen</td><td>1031873</td><td></td></tr><tr><td>LightCycler centrifuge</td><td>Roche</td><td>75005087</td><td></td></tr><tr><td>LightCycler Cooling Block</td><td>Roche</td><td>10800058001</td><td></td></tr><tr><td>LightCycler Capping Tool</td><td>Roche</td><td>3357317</td><td></td></tr><tr><td>Color Compensation Kit</td><td>Roche</td><td>2158850</td><td></td></tr><tr><td>LightCycler 20 &amp;mu;l Capillaries</td><td>Roche</td><td>1 909 339</td><td></td></tr><tr><td>Blades</td><td>Sakura</td><td>Fisher Scientific: 4689</td><td>Accu-Edge low profile</td></tr></tbody></table>

qpcr is a sensitive and rapid method for detection of cytomegaloviral dna in formalin-fixed, paraffin-embedded biopsy tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

A total of 228 tissue blocks were tested by quantitative polymerase chain reaction (qPCR), which was comprised of 91 cytomegalovirus (CMV) positive cases based on histology and positive immunohistochemistry (IHC), 18 with equivocal CMV IHC, and 79 negative controls. As illustrated in Figure 2, CMV positive cases would have demonstrated typical CMV viral inclusions (A) and/or positive IHC staining (B). Equivocal cases would have demonstrated rare, non-classic appearing st...

Watch this Scientific Journal Video about qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue at JoVE.com

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

qpcr-is-sensitive-rapid-method-for-detection-cytomegaloviral-dna

Research

JoVE Journal

Biology

15.7K Views.  Indiana University School of Medicine. This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

Video: qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

Immunology and Infection

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its excellent preservation of morphology. Personalized medicine increasingly provides individually adapted and targeted therapies for characterized individual diseases enabled by combined morphological and molecular analytical technologies and diagnostics. Performance of morphologic and molecular assays from the same FFPE specimen is challenging because of the negative impact of formalin due to chemical modification and cross-linking of nucleic acids and proteins. A non-cross-linking, formalin-free tissue fixative has been recently developed to fulfil both requirements, i.e., to preserve morphology like FFPE and biomolecules like cryo-preservation. Since FISH is often required in combination with histopathology and molecular diagnostics, we tested the applicability of FISH protocols on tissues treated with this new fixative. We found that formalin post-fixation of histological sections of non-cross-linking, formalin-free and paraffin-embedded (NCFPE) breast cancer tissue generated equivalent results to those with FFPE tissue in human epidermal growth factor receptor 2 (HER2) FISH analysis. This protocol describes how a FISH assay originally developed and validated for FFPE tissue can be used for NCFPE tissues by a simple post-fixation step of histological sections.

Fluorescence in-situ hybridization (FISH) is often required in combination with histopathology and molecular diagnostics for selection of therapy in personalized medicine. A novel non-cross-linking, formalin-free tissue fixative that allows high quality morphologic, molecular and FISH analyses from the same specimen by addition of a post-fixation step before FISH is presented.

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its ...

Cancer Research

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 ...

Genetics

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Neuroscience

Enhancing FISH Analysis for Oncogenic Drivers in Cancer Tissue Samples

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While sequencing-based methodologies enable an unbiased identification of ecDNA, cytogenetic-based techniques, such as fluorescence in situ hybridization (FISH), remain time and cost-effective for identifying ecDNA in clinical specimens. The application of FISH in formalin-fixed paraffin-embedded (FFPE) tissue samples offers a unique avenue for detecting amplified genes, particularly when viable specimens are not available for karyotype examination. However, there is a lack of consensus procedures for this technique. This protocol provides comprehensive, fully optimized, step-by-step instructions for conducting FISH to detect gene amplification, including ecDNA, in FFPE tissue samples which present unique challenges that this protocol aims to overcome and standardize. By following this protocol, researchers can reproducibly acquire high-quality imaging data to assess gene amplification.

Author Spotlight: FISH as a Tool for Precise Gene Amplification Assessment in Cancer Specimens

This protocol provides a reproducible method to visualize gene amplification in formalin-fixed paraffin-embedded (FFPE) tissue specimens.

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While ...

Merkel Cell Polyomavirus Infection and Detection

Merkel cell polyomavirus (MCPyV) infection can lead to Merkel cell carcinoma (MCC), a highly aggressive form of skin cancer. Mechanistic studies to fully investigate MCPyV molecular biology and oncogenic mechanisms have been hampered by a lack of adequate cell culture models. Here, we describe a set of protocols for performing and detecting MCPyV infection of primary human skin cells. The protocols describe the isolation of human dermal fibroblasts, preparation of recombinant MCPyV virions, and detection of virus infection by both immunofluorescent (IF) staining and in situ DNA-hybridization chain reaction (HCR), which is a highly sensitive fluorescence in situ hybridization (FISH) approach. The protocols herein can be adapted by interested researchers to identify other cell types or cell lines that support MCPyV infection. The described FISH approach could also be adapted for detecting low levels of viral DNAs present in the infected human skin.

Here, we present a protocol to infect primary human dermal fibroblast with MCPyV. The protocol includes isolation of dermal fibroblasts, preparation of MCPyV virions, virus infection, immunofluorescence staining, and fluorescence in situ hybridization. This protocol can be extended for characterizing MCPyV-host interactions and discovering other cell types infectable by MCPyV.

Merkel cell polyomavirus (MCPyV) infection can lead to Merkel cell carcinoma (MCC), a highly aggressive form of skin cancer. Mechanistic studies to fully ...

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.

The goal of this video is to demonstrate how to perform&#160;automated DNA extraction from formalin-fixed paraffin-embedded (FFPE) reference standard cell lines and digital droplet PCR&#160;(ddPCR) analysis to detect rare mutations in a clinical setting. Detecting mutations in FFPE samples demonstrates the clinical utility of ddPCR in FFPE samples.

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

Summary

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Chapters in this video

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

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

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization

Merkel Cell Polyomavirus Infection and Detection

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry