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 border="0" cellpadding="0" cellspacing="0" width="745">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">1.7 mL microfuge tubes</td>
			<td style="width:195px;">Costar</td>
			<td align="right" style="width:160px;">3620</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">serrated forceps</td>
			<td style="width:195px;">Electron Microscopy Services</td>
			<td style="width:160px;">62086-1S</td>
			<td>Micro Forceps MF-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Tabletop centrifuges (microfuge)</td>
			<td style="width:195px;">Eppendorf</td>
			<td align="right" style="width:160px;">5424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Xylene</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:160px;">Fisher Scientific: X3P</td>
			<td>1 gallon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Ethanol</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:160px;">Fisher Scientific: ET108</td>
			<td>96-100%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">microtome</td>
			<td style="width:195px;">Leica</td>
			<td style="width:160px;">RM2255</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">QIAamp DNA FFPE Tissue Kit</td>
			<td style="width:195px;">Qiagen</td>
			<td align="right" style="width:160px;">56404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">artus CMV LightCycler PCR ASR</td>
			<td style="width:195px;">Qiagen</td>
			<td align="right" style="width:160px;">4500025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">CMV LC PCR Supplement Kit</td>
			<td style="width:195px;">Qiagen</td>
			<td align="right" style="width:160px;">1031873</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">LightCycler centrifuge</td>
			<td style="width:195px;">Roche</td>
			<td align="right" style="width:160px;">75005087</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">LightCycler Cooling Block</td>
			<td style="width:195px;">Roche</td>
			<td align="right" style="width:160px;">10800058001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">LightCycler Capping Tool</td>
			<td style="width:195px;">Roche</td>
			<td align="right" style="width:160px;">3357317</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Color Compensation Kit</td>
			<td style="width:195px;">Roche</td>
			<td align="right" style="width:160px;">2158850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">LightCycler 20 &#956;L Capillaries</td>
			<td style="width:195px;">Roche</td>
			<td style="width:160px;">1 909 339</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">blades</td>
			<td style="width:195px;">Sakura</td>
			<td style="width:160px;">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

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

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 ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

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

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

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

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