This research was supported by a team grant from Movember/Prostate Cancer Canada to JMSB, DMB, PCP, and JL, and by the Ontario Institute of Cancer Research (JMSB, DMB, and PCP) and Motorcycle Ride for Dad Kingston/University Hospitals Kingston Foundation/Kingston General Hospital (DMB, PCP).

1. Tissue Coring
<ol>
	<li>Review the microscope slide and outline the region(s) of interest using a fine-point permanent marker. Cut out a section of paraffin film large enough to cover the region of interest on the microscope slide. Place film firmly on slide and wrap film over edges to keep the film from slipping. Using a fine-point permanent marker, outline the entire tissue and the region(s) of interest within the tissue, keeping the outline touching&#160;&#8211; but outside of &#8211; the region(s).</li>
	<li>Remove the film and transfer it to the corresponding tissue block. Orient the film by flipping or rotating it so that the outline of the entire tissue matches the observed shape of the tissue in the block (Figure 1). Press the section of film firmly to the surface of the block to prevent slippage.</li>
	<li>Using the tip of the permanent marker, make shallow but visible (~0.2 mm) indentations along the outline of the region(s) of interest, then remove the film. Load 1 ml of bleach, 70% ethanol, and water into separate 1.5 or 2.0 ml microcentrifuge tubes.</li>
	<li>Clean the receptor (red) punch from the 0.6 mm punch set by sliding the punch up and down several times while the tip is submerged into the tube containing bleach. Repeat the above step with 70% ethanol and then water (critical to ensure that bleach is removed).</li>
	<li>Press the punch into the tissue, inside the region of interest to a depth of 3 mm and withdraw the punch. Release the core into a low binding 1.5 or 2 ml tube by pushing it out of the punch with the stylus. Store the cores at -20 &#176;C (long-term) or 4 &#176;C for short-term use.</li>
	<li>Clean the punch according to step 1.4 and continue with the next regions or sample.</li>
</ol>
2. Deparaffinize the FFPE Tissue Cores
<ol>
	<li>Carryout deparaffinization in 1.5 or 2 ml tubes by adding 1 ml xylene to the tissue core and vortexing vigorously for 10 sec. Heat for 3 min at 50 &#176;C.</li>
	<li>Centrifuge for 2 min at room temperature (RT) and maximum speed (21,130 x g) and place tube on ice for 5 min (allows the waxy residue to solidify on the top).</li>
	<li>Carefully remove paraffin accumulated around meniscus with supernatant using a pipette tip and repeat xylene treatment (steps 2.1-2.2).</li>
	<li>Add 1 ml of ethanol (100%) and vortex vigorously for 10 sec. Centrifuge for 2 min at RT (maximum speed), and carefully discard the ethanol. Repeat the above step once.</li>
</ol>
3. Homogenization of the Deparaffinized Cores
<ol>
	<li>Resuspend the cores in 700 &#181;l of ethanol (100%) prior to homogenization. Using a motorized tissue homogenizer, grind the cores into fine tissue particles (~1 min on medium setting). Clean the homogenizer probe between each sample to minimize carry-over contamination.
	<ol>
		<li>Fill 15 ml tubes with ~10 ml of bleach, RNase neutralizing solution and 70% ethanol. After sample homogenization, wash the homogenizer probe in each of the cleaning solutions in the order stated above. Run the homogenizer on the highest speed during the washing stage.</li>
		<li>Wipe the probe with tissue and allow probe to dry completely before homogenizing the next sample. Visually inspect the probe blades for residual tissue pieces. If found, clean the probe again. Change the cleaning solutions (bleach, ethanol, and RNase neutralizing solution) daily.</li>
	</ol>
	</li>
	<li>Following homogenization, bring the sample volume to 1 ml by adding more 100% ethanol (~300 &#181;l). Centrifuge at maximum speed for 15 min, carefully aspirate the ethanol and air dry pellet for approximately 15-20 min before proceeding with RNA extraction.</li>
</ol>
4. Digestion with Proteinase K
<ol>
	<li>Resuspend the pellet in 150 &#181;l Proteinase K digestion Buffer and flick tube to loosen the pellet. Add 10 &#181;l of temperature-stable proteinase K and mix by flicking (do not vortex the tube). Incubate the content in the tube at 56 &#176;C for 15 min with mild agitation.</li>
	<li>Allow tube to incubate on ice for 3 min. Complete cooling is important for efficient precipitation in the following step. Centrifuge for 15 min at maximum speed.</li>
</ol>
5. Separate RNA from DNA
<ol>
	<li>Carefully transfer the supernatant, without disturbing the pellet, to a new 1.5 ml for RNA purification.</li>
	<li>Keep the pellet for DNA purification (pellet can be stored for 2 hr at RT, for up to 1 day at 2-8 &#176;C, or for longer periods at -20 &#176;C).</li>
</ol>
6. RNA Purification
<ol>
	<li>Incubate the RNA-containing supernatant at 80 &#176;C for 15 min (do not exceed this time). Next, briefly centrifuge the tube to collect drops from the inside of the lid.</li>
	<li>Add 320 &#181;l Buffer RLT to adjust binding conditions, and mix by pipetting. Next, add 720 &#181;l ethanol (100%), and vortex.</li>
	<li>Transfer 600 &#181;l of the sample, including any precipitate that may have formed, to RNA spin column (supplied in the kit) placed in a 2 ml collection tube and set aside the remaining content. Centrifuge for 15 sec at &#8805;8,000 x g, discard the flow-through and reuse the collection tube.</li>
	<li>Transfer remaining sample onto a column, including droplets that may have accumulated in the lid of the tube, centrifuge for 15 sec at &#8805;8,000 x g, and discard the flow-through.</li>
	<li>Add 350 &#181;l Buffer FRN to the spin column and centrifuge for 15 sec at &#8805;8,000 x g, discard the flow-through and reuse collection tube.</li>
	<li>Gently mix 10 &#181;l DNase I stock solution with 70 &#181;l Buffer RDD, add directly to the spin column membrane, and incubate at RT for 15 min.</li>
	<li>Add 500 &#181;l Buffer FRN to the spin column, centrifuge for 15 sec at &#8805;8,000 x g and save the flow-through for use in the next step. To enhance recovery of small RNAs, place the spin column in a new 2 ml collection tube and apply the flow-through from the previous step to the spin column.</li>
	<li>Centrifuge for 15 sec at &#8805;8,000 x g, discard the flow-through and reuse the collection tube in next step. Add 500 &#181;l Buffer RPE to the spin column and centrifuge for 15 sec at &#8805;8,000 x g, discard the flow-through and reuse the collection tube in the next step.</li>
	<li>Add 500 &#181;l Buffer RPE to the spin column and centrifuge for 15 sec at &#8805;8,000 x g and discard the collection tube with the flow-through.</li>
	<li>Place the spin column in a new 2 ml collection tube, open the lid and centrifuge at maximum speed for 5 min. Discard the collection tube with the flow-through.</li>
	<li>Place the spin column in a new 1.5 ml collection, add 20 &#181;l of RNase-free water directly onto the spin column membrane, and incubate the tube for 1 min at RT. Centrifuge at maximum speed for 1 min to elute the RNA. Store the eluted RNA sample at -80 &#176;C.</li>
</ol>
7. DNA Purification
<ol>
	<li>Resuspend the pellet obtained during the RNA extraction by stepwise addition of 45 &#181;l of proteinase K buffer (400 mM Tris 7.5, 400 mM NaCl, 3 mM MgCl2, 4% SDS); 45 &#181;l H2O; and 400 &#181;g of high potency Proteinase K.</li>
	<li>Incubate the above solution at 56 &#176;C for 24 hr (recommended) or overnight. Performincubation at 90 &#176;C for 2 hr without agitation and briefly centrifuge the microcentrifuge tube to collect drops from inside of the lid.</li>
	<li>Allow the sample to cool to RT and then add 4 &#181;l RNase A (100 mg/ml). Incubate the sample for 2 min at RT.</li>
	<li>Add 200 &#181;l Buffer AL to the sample, and mix thoroughly by vortexing. Next, add 200 &#181;l of 100% ethanol, and mix thoroughly by vortexing. Transfer the entire sample to the provided spin column, place in a 2 ml collection tube, and centrifuge for 1 min at &#8805;8,000 x g.</li>
	<li>Discard the collection tube with the flow-through and place the spin column in a new 2 ml collection tube. Add 700 &#181;l Buffer AW1 to the spin column, centrifuge for 15 sec at &#8805;8,000 x g, discard the flow-through and reuse the collection tube.</li>
	<li>Add 700 &#181;l Buffer AW2 to the spin column, centrifuge for 15 sec at &#8805;8,000 x g, discard the flow-through and reuse the collection tube. Next, add 700 &#181;l of 100% ethanol to the spin column, centrifuge for 15 sec at &#8805;8,000 x g and discard the collection tube with the flow-through.</li>
	<li>Place the spin column in a new 2 ml collection tube, open the lid of the spin column, and centrifuge at full speed for 5 min. Discard the collection tube with the flow-through.</li>
	<li>Place the spin column in a new 1.5 ml collection tube and add 25 &#181;l of heated nuclease-free water (50 &#176;C). Incubate column and tube at 50 &#176;C for 10 min. Centrifuge for 1 min at maximum speed, add 25 &#181;l of nuclease-free water (RT) to column and incubate for 1 min at RT.</li>
	<li>Centrifuge for 1 min at maximum speed (21,130 x g) and harvest flow-through containing genomic DNA (approximately 50 &#181;l of DNA in total). Store the column at -20 &#176;C (in case another elution is required later).</li>
</ol>

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Histopathol. 26 (6), 797-810 (2011).</li><li>Beltran, 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=Targeted+Next-generation+Sequencing+of+Advanced+Prostate+Cancer+Identifies+Potential+Therapeutic+Targets+and+Disease+Heterogeneity.">Targeted Next-generation Sequencing of Advanced Prostate Cancer Identifies Potential Therapeutic Targets and Disease Heterogeneity.</a> Eur. Urol. 63 (5), 920-926 (2013).</li><li>Hoppin, J. A., Tolbert, P. E., Taylor, J. A., Schroeder, J. C., Holly, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+for+selection+bias+with+tumor+tissue+retrieval+in+molecular+epidemiology+studies.">Potential for selection bias with tumor tissue retrieval in molecular epidemiology studies.</a> Ann. 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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=Comparison+of+methods+in+the+recovery+of+nucleic+acids+from+archival+formalin-fixed+paraffin-embedded+autopsy+tissues.">Comparison of methods in the recovery of nucleic acids from archival formalin-fixed paraffin-embedded autopsy tissues.</a> Anal. Bochem. 400 (1), 110-117 (2010).</li><li>Abramovitz, 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=Optimization+of+RNA+extraction+from+FFPE+tissues+for+expression+profiling+in+the+DASL+assay.">Optimization of RNA extraction from FFPE tissues for expression profiling in the DASL assay.</a> BioTechniques. 44 (3), 417-423 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> AllPrep DNA/RNA FFPE Handbook. , (2012).</li><li>Laurent, L. 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The authors declare no competing financial interests.

For successful extraction of DNA and RNA from tissue regions of interest, accurate coring is critical. This protocol describes the use of a tissue punch to isolate 0.6 mm diameter cores and outlines the process for transferring notations from microscope slides to corresponding FFPET blocks. Modifications to the manufacturer&#39;s protocol were required to efficiently extract nucleic acids from cores, which are approximately 50 times thicker than the microtome sections for which the protocol was intended. Since the cores may contain more paraffin wax relative to tissue sections, effective deparaffinization of cores through repeated xylene and ethanol treatment steps were required. The success of the post-deparaffinization steps depended on proper mechanical tissue cores homogenization and efficient proteinase K digestion. Further optimization of the proteinase K digestion can be performed.
It is worth mentioning that this method identifies areas of interest on the surface of the block, as identified in corresponding histopathology slides. As the core harvests tissue that may be 3 or 4 mm deep, users of this protocol may be concerned about what cells or tissues lay beneath the block surface. While this is a valid concern, multiple studies (reviewed in reference27) have demonstrated that tissue cores faithfully represent the histologic and molecular features of pathologic tissue blocks, particularly when duplicate or triplicate cores are sampled from the area of interest.
As the modified commercial extraction kit adopted in this protocol enables concurrent extraction of both DNA and RNA from the same tissue, the protocol saves precious biological material and allows a direct comparison between the two resulting nucleic acids from the same sample. Concurrent extraction of RNA and DNA cuts down labor and tissue depletion by half, and enables precise integrated analysis of gene expression, as well as epigenetic and genetic features found in DNA. Since the yields of both RNA and DNA from these representative tissue cores typically exceed 600 and 300 ng, respectively, and since most current PCR and next generation sequencing applications typically require 10-100 ng, most of the samples purified by this protocol should provide adequate material for several downstream assays. This protocol has been shown to be reproducible across independent laboratories (Selvarajah et al., In Prep.). RNA from this protocol was of sufficient quality for gene expression analysis using either RT-PCR or a popular multianalyte platform, and DNA performed well in methylation specific PCR assays. Future studies aimed at assessing the utility of recovered nucleic acids in next generation sequencing are warranted.
Thus, several modifications were made to a commercially available protocol, designed for thin FFPET sections, rendering it suitable for the co-extraction of RNA and DNA from 0.6 mm FFPET cores. The protocol demonstrated consistently high yields in a large cohort of prostate cancer samples and in a limited set of samples from cancers of the breast, brain and bladder. Overall, the protocol should enable users to carry out targeted gene-based analyses of large well-annotated tissue collections. Importantly, the protocol enables efficient focused sampling of regions of interest in FFPET, relatively little hands-on time, and high enough yields for most downstream applications.

Genomic biomarker research seeks to identify molecular correlates that accurately and reliably reflect disease status, and do so in a clinically useful manner.1 Biomarker development is reliant on retrospective analysis of well-annotated tissue samples. Diseased and normal tissue samples are stored either as fresh-frozen tissue in specialized biobanks or as formalin-fixed paraffin-embedded tissue (FFPET) blocks in clinical archives. Fresh-frozen tissue allows for the extraction of high-quality nucleic acids and has been widely used in genomic biomarker discovery studies.2,3 However, fewer tissue samples are available in biobanks and studying such tissue introduces a bias towards larger samples, unusual categories of disease, and patients seen at specialized centers with greater abilities to bank tissue.4 FFPET, in contrast, is the default storage method for diseased human and animal tissues. While FFPET blocks maintain cellular morphology, the fixation process cross-links other cellular constituents to nucleic acids. Cross-linked RNA and DNA are recoverable, but only in degraded, highly fragmented forms.5,6 However, these DNA and RNA fragments are amenable to analysis by an expanding array of assays, including mRNA expression, DNA hypermethylation, and targeted sequencing.7,8 To exploit this opportunity in the large quantity and variety of FFPET available for research, there is a need for an efficient and reliable extraction protocol.
A large proportion of biomarker research in tissue focuses on cancer. Like other types of diseased tissue, cancer tissue often shows significant regional heterogeneity in cell preservation and cell type. Since biomarker research relies on the ability to correlate constituents of diseased tissue with molecular features, a critical step of this process is the precise harvesting of tissue that is well preserved and enriched for the disease under study. In FFPET, two enrichment techniques are often utilized: laser capture microdissection (LCM), and microtome sectioning. LCM enables highly focused tissue harvesting and can be used to isolate specific, well-preserved cell types in heterogeneous tissues.9,10 However, LCM requires expensive equipment and is prohibitively time consuming for large numbers of samples. Microtome sectioning is a more widely-used process where thin sections are cut from FFPET blocks.11,12 Microtome-cut sections often include tissue that is heterogeneous in cell preservation (e.g., necrotic vs. well-preserved) and composition (e.g., cancer vs. benign parenchyma), and hence may lead to the homogenization of molecular features best investigated separately. Thus, there is a need for a high throughput method that enriches for cells of interest. A third method, isolation of nucleic acids from FFPET cores, provides this enrichment, is suitable for high throughput protocols, and has been used by others to isolate RNA or DNA from separate tissue cores.7,13,14
A number of published protocols specify methods of extracting nucleic acids from FFPET (Table 1). However, protocols where RNA and DNA are extracted from the same tissue have been optimized for microtome tissue sections, but not for tissue cores.15,16 Similarly, published protocols which offer increased tissue specificity, either through tissue cores or slide microdissections, specify procedures for extraction of DNA, but not RNA.7,17 Here, an optimized protocol for dual extraction of both DNA and RNA from the same tissue core is demonstrated. Tissue cores are harvested by inserting tissue microarray (TMA) punches into regions of interest mapped onto FFPET blocks. The mapping is performed by annotating a microscope slide with a marker pen and transferring the annotation to the surface of the corresponding FFPET block (Figure 1).
Prior work that led to the development of this protocol included a comparison of several commercially available nucleic acid extraction systems. In this comparison, modifications to commercial protocols, as described below provided the highest DNA and RNA yields and quality (Selvarajah et al., In Prep). Tissue cores are thicker than the 5-10 &#181;m micron sections typically used in FFPET extraction protocols11,12,14,18-20, and may contain more variable amounts of paraffin. To compensate for this, deparaffinization was enhanced by repeating xylene and ethanol treatments and by introducing a motorized homogenization step (Figure 1). Furthermore, proteinase K digestion times were lengthened to increase DNA yield. Overall, this protocol is cost-effective and enables the establishment of linkages between molecular and histopathologic features of disease in large, well characterized populations. The protocol in its entirety can be carried out reliably within 2 days, including 3 hr of hands-on time, with little need for specialized or expensive equipment.
The step-by-step protocol is hereafter as a modified version of the manufacturer&#39;s protocol.21 Please see Table of Materials/Equipment for specific reagents, equipment, and manufacturers.

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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Plastic paraffin film, &#34;Parafilm &#39;M&#39;&#34;</td>
			<td style="width:217px;">Bemis&#160;</td>
			<td style="width:119px;">RK-06720-40</td>
			<td style="width:167px;">Any generic paraffin film will work as a substitute</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Sodium Hypochlorite, &#34;Ultra Bleach&#34;</td>
			<td style="width:217px;">Likewise</td>
			<td style="width:119px;">53-2879-2</td>
			<td>Any generic bleach will work as a substitute. Hazardous material that can cause burns on contact.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Molecular biology grade absolute ethanol</td>
			<td style="width:217px;">Fisher BioReagents</td>
			<td style="width:119px;">BP2818-500</td>
			<td>Sigma-Aldrich E7023 suffices as a substitute</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Molecular Grade H2O</td>
			<td style="width:217px;">G-Biosciences</td>
			<td style="width:119px;">786-293</td>
			<td>Sigma W4502 suffices, as well as any other brand of molecular grade H2O</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">0.6mm Punch Set for Beecher Instruments&#160;</td>
			<td style="width:217px;">Estigen</td>
			<td style="width:119px;">MPO6[Yellow]</td>
			<td>Make sure to use the red receiver punch from the set</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Fine point permanent marker</td>
			<td style="width:217px;">Sharpie</td>
			<td style="width:119px;">10365796S</td>
			<td>Using the marker on FFPE tissues causes it to dry out quickly, so several may be required</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">FFPE tissue block</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Stained tissue slide corresponding to FFPE block</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">1.5 mL Micro-Centrifuge Tubes</td>
			<td style="width:217px;">Fisher BioReagents</td>
			<td style="width:119px;">05-408-137</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">2.0 mL Low binding tubes (LoBind Micro-Centrifuge Tubes)</td>
			<td style="width:217px;">Eppendorf</td>
			<td style="width:119px;">22431048</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">1.5 mL Low binding tubes (LoBind Micro-Centrifuge Tubes)</td>
			<td style="width:217px;">Eppendorf</td>
			<td style="width:119px;">22431021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Histology Xylene</td>
			<td style="width:217px;">VWR</td>
			<td style="width:119px;">CA 95057-822</td>
			<td>Fisher Scientific X5-500 suffices as a substitute</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Molecular Biology Grade 2-Propanol</td>
			<td style="width:217px;">Sigma</td>
			<td style="width:119px;">I9516</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:283px;">AllPrep FFPE DNA/RNA Kit&#160;</td>
			<td style="width:217px;">Qiagen</td>
			<td style="width:119px;">80234</td>
			<td>Prepare buffers accodring to the AllPrep DNA/RNA FFPE Handbook21</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Buffers: RLT, FRN, RPE, ATL, AL, AW1, AW2, DNaseI solution</td>
			<td style="width:217px;">Qiagen</td>
			<td style="width:119px;">80234</td>
			<td>Prepare buffers accodring to the AllPrep DNA/RNA FFPE Handbook21</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Temperature stable proteinase K</td>
			<td style="width:217px;">Qiagen</td>
			<td style="width:119px;">80234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">High potency proteinase K</td>
			<td style="width:217px;">Invitrogen</td>
			<td style="width:119px;">25530-049</td>
			<td>Invitrogen 25530-015 suffices as a substitute</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAse neutralizing solution (Rnase AWAY)</td>
			<td style="width:217px;">Molecular BioProducts</td>
			<td style="width:119px;">7003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">RNaseA 100mg/mL</td>
			<td style="width:217px;">Qiagen</td>
			<td style="width:119px;">19101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">BD Integra Syringe 3mL 21G x 1/2</td>
			<td style="width:217px;">BD&#160;</td>
			<td style="width:119px;">305274</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Motorized tissue homogenizer (TissueRuptor)</td>
			<td style="width:217px;">Qiagen&#160;</td>
			<td style="width:119px;">9001271</td>
			<td>Fisher Scientific 14-261-29 suffices as a substitute</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">-20&#176;C and -80&#176;C Laboratory Freezer &#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Micro-Centrifuge with rotor for 2mL tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Digital Vortex Mixer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Pipettes and filter tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Heating blocks or water baths</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Tris Hydrochloride&#160;</td>
			<td style="width:217px;">Amresco</td>
			<td style="width:119px;">0234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Sodium Chloride&#160;</td>
			<td style="width:217px;">Amresco</td>
			<td style="width:119px;">0241</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Anhydrous Magnesium Chloride</td>
			<td style="width:217px;">Sigma</td>
			<td style="width:119px;">M8266</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Sodium Dodecyl Sulfate</td>
			<td style="width:217px;">Sigma</td>
			<td style="width:119px;">L4509</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Acrodisc 25mm syring filters with 0.45 um Supor membrane</td>
			<td style="width:217px;">Pall</td>
			<td style="width:119px;">PN 4614</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Syringe with retracting BD PrecisionGlide needle 3mL</td>
			<td style="width:217px;">BD Integra</td>
			<td style="width:119px;">305274</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Hydrochloric Acid</td>
			<td style="width:217px;">BDH</td>
			<td style="width:119px;">3026</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:283px;">Multianalyte gene expression platfrom (nCounter &#174; CAE codeset and Nanostring nCounter platform)</td>
			<td style="width:217px;">Nanostring nCounter platform, Nanostring</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:283px;">Fluorometric nucleic acid quantification (Qubit dsDNA HS Assay Kit and Qubit&#174; RNA BR Assay Kit)</td>
			<td style="width:217px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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 protocol represents an optimized method for recovering DNA and RNA from tissue cores, using modifications of a commercial extraction system designed for tissue sections. Optimization included the introduction of tissue homogenization, utilization of more potent Proteinase K for DNA extraction, and extension of tissue digestion time. Graphs and statistical analyses included 2-way ANOVA, linear regression and correlation.
Optimizations of Proteinase Digestion 
The commercial kit included a room temperature stable proteinase K solution which was substituted with a more potent proteinase K, resulting in higher DNA yield (Figure 2A). To further increase the DNA yields, digestion was extended from 2 to 24 hr. No significant differences were seen between the two time points, but the 24 hr digestion appeared to provide more consistent yields across samples. However, further incubation to 48 hr did not further improve DNA recovery (Figure 2B; p = 0.74).
Typical DNA Recovery from FFPE Prostate Cancer Tissue Samples
Using the optimized protocol, RNA and DNA were co-extracted from 333 prostate cancer FFPET samples ranging from 3 to 14 years in sample age. From each sample, 3 tissue cores (average total tissue volume of 0.95 &#177; 0.13 mm3) were used as input. While there are other microfluidic based gel-electrophoresis methods which can estimate concentrations and provide evaluations of the size distribution of nucleic acids molecules, such methods do not provide reproducible nucleic acids quantification, and cannot distinguish between RNA and DNA as flourometrically-based assays do.22 And, because microfluidic based gel-electrophoresis results are not reliable for fragmented nucleic acids derived from FFPET,23 nucleic acid yields were measured fluorometrically (see reagent list for details). The average yield was 2,270 ng of RNA and 820 ng of DNA (Figure 3A). Approximately 90% of all FFPET samples analysed in this study yielded &#8805;100 ng of DNA and &#8805; 500 ng of RNA. Interestingly, there was no significant correlation between the age of the FFPET sample and nucleic acid recovery (Figure 3B). Overall, RNA and DNA yields were correlated across samples (R2 = 0.39; p &#60;0.0001), although more than twice as much RNA than DNA was recovered from each sample (Figure 3C).
As the pilot and optimization work was performed on prostate tissues, the next step was to investigate the performance of this protocol on a few additional types of archival tissue. Starting with surgically removed and autopsy FFPET samples representing benign liver (1 sample from 1 case), cancers of the brain (8 samples from 1 case), urinary bladder (2 samples from 2 cases), and breast (3 samples from 3 cases), the protocol yielded &#62;100 ng of DNA and RNA from 90% of samples (Figure 3D). While nucleic acid yields were lower in autopsy tissues than in surgical tissues, representative results indicate that the protocol produces similar yields across cancers derived from different sites.
Assessment of RNA and DNA Integrity and their Representative Performance in Downstream Analysis
RNA expression analysis of 47 genes in 8 selected FFPET prostate cancer samples and a fresh PC-3 prostate cancer cell line sample (as a positive control) was performed using a commercial multianalyte gene expression platform that is optimized for FFPET. The mRNA counts in PC3 were typically higher than those from FFPET samples (Figure 4A). However, comparing relative expression of all genes, FFPET prostate cancer samples showed similar expression profiles to PC-3 RNA, indicating that both sources of RNA are suitable for RNA expression profiling.
To demonstrate performance of genomic DNA extracted with this protocol, bisulfite-converted DNA extracts from FFPET samples were amplified by methylation specific PCR (MSP).24 MSP analysis of ALU repetitive elements, highly methylated regions present in millions of copies in the human genome,25 was used as a genomic methylation control, and expected to show minimal variations between samples. As shown in Figure 4B, there was little to no variation seen between different samples in ALU MSP methylation levels. Further, MSP assays based on GSTP1, a gene known to be hypermethylated in prostate cancer but not in benign samples,26 showed no detectable amplifications in DNA from benign samples. As expected, lower qPCR cycle threshold values were detected in DNA from cancer tissues, indicating enrichment of methylated GSTP1 copies. The utility of nucleic acids recovered by this protocol was further tested in typical downstream assays, using nucleic acids recovered from benign liver and from a brain (post-mortem) and from two surgically removed breast cancer samples. Both RT-qPCR based expression and MSP assays performed well on breast cancer and liver FFPET, but the RT-PCR assay failed to amplify a highly expressed mRNA from the post-mortem brain tumor sample (Figure 4C), suggesting that RNA had degraded, likely due to delayed tissue fixation.
<img alt="Figure 1" src="/files/ftp_upload/54299/54299fig1.jpg" /> 
Figure 1: Overview of the Extraction Procedure for FFPET Samples. The figure illustrates how an area of interest in a tissue block is mapped based on histopathologic selection from a microscope slide. Three 0.6 mm tissue cores are then obtained from each tissue area by using biopsy punches, homogenized together and then subjected to extraction of both RNA and DNA. <a href="https://www.jove.com/files/ftp_upload/54299/54299fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54299/54299fig2.jpg" /> 
Figure 2: Nucleic Acids (DNA and RNA) yields in ng/mm3 of FFPET from two Proteinase K (Temperature Stable and High Potency Enzymes), and Tested across a Range of Incubation Times for the Latter. (A) Performance of Proteinase K from different suppliers. DNA extractions were performed on a representative FFPET sample using temperature stable enzyme supplied with the kit versus a more potent enzyme from another manufacturer. (B) Determining optimum Proteinase K incubation time to maximize the DNA yield. Performance of high concentration Proteinase K digestion was evaluated at three different incubation periods using 3 FFPET samples. Error bars represent standard error of mean (SEM). <a href="https://www.jove.com/files/ftp_upload/54299/54299fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54299/54299fig3.jpg" /> 
Figure 3: Nucleic Acids (DNA and RNA) yields in ng/mm3 of FFPET in Total, across Sample Years and Representative Tissue Types. (A) Total recovered nucleic acids from formalin-fixed paraffin-embedded tissue. The nucleic acids quantities presented are based on the extractions of 333 FFPET samples using the optimized protocol. (B) Correlation plot between recovered total DNA and RNA and the age of FFPET samples. The extracted FFPET samples used were obtained from the years 2000 to 2012. (C) Correlation between yields from concurrently extracted DNA and RNA from 333 prostate samples. There is a positive correlation between DNA and RNA yields. (D) Demonstration of the protocol using additional archival tissue types. The optimized protocol was used to extract nucleic acids from 14 cancer (breast, bladder and brain) and normal (liver) samples. Error bars represent SEM. <a href="https://www.jove.com/files/ftp_upload/54299/54299fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54299/54299fig4.jpg" /> 
Figure 4: Performance of RNA and DNA Co-extracted from Tissue Cores in Downstream Applications. (A) mRNA counts for FFPE prostate cancer tissues and for fresh PC-3 cell line control. Each point represents the average of 3 technical replicates extracted separately. Fresh PC-3 cell line RNA values are illustrated by yellow bars and FFPET tissue values are represented by coloured dots. (B) Methylation specific PCR assays on DNA of FFPET prostate cancer samples. Cycle threshold values were obtained for 10 samples performed as expected using 50 ng/reaction of bisulfite converted DNA. ALU MSP assays from all FFPET samples had similar cycle threshold values (p &#62;0.67). GSTP1 MSP assays showed higher methylation (lower cycle threshold) levels in prostate cancer than in benign prostate. (C) Assessment of DNA and RNA quality from additional tissue types. HPRT1 gene expression and Alu gene methylation assays were performed on nucleic acids (RNA and DNA respectively) extracted from normal liver (autopsy), and brain (autopsy) and breast cancers (all FFPET). Results from prostate are shown for comparison. Note: similar results were observed from each tissue type, except for failed mRNA amplification from one autopsy sample. Each point or bar represents a sample, and error bars represent SEM. <a href="https://www.jove.com/files/ftp_upload/54299/54299fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="12">Tissue Cores</td>
		</tr>
		<tr>
			<td rowspan="2"></td>
			<td rowspan="2">Tissue Input (mm3)</td>
			<td rowspan="2">Extracted Nucleic Acid</td>
			<td colspan="2">Validation</td>
			<td colspan="3">Quality check: DNA</td>
			<td colspan="4">Quality check: RNA</td>
		</tr>
		<tr>
			<td>(# of samples)</td>
			<td>Age of Sample (years)</td>
			<td>Total Yield (ng)</td>
			<td>Fragment size (bp)</td>
			<td>PCR</td>
			<td>Total Yield (ng)</td>
			<td>Fragment size (bp)</td>
			<td>PCR</td>
			<td>NanoString</td>
		</tr>
		<tr>
			<td>Pikor et al.&#160;</td>
			<td>43 - 129</td>
			<td>DNA</td>
			<td>No data</td>
			<td>No data</td>
			<td>No data</td>
			<td>No data</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Montaser-Kouhsari et al.</td>
			<td>18 - 29.5</td>
			<td>RNA</td>
			<td>763</td>
			<td>0-25</td>
			<td></td>
			<td></td>
			<td></td>
			<td>843</td>
			<td>No data</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>This paper</td>
			<td>1.71</td>
			<td>DNA and RNA</td>
			<td>&#62;350</td>
			<td>3-12</td>
			<td>820</td>
			<td>&#160;100 - 500</td>
			<td><img alt="Check Mark" src="/files/ftp_upload/54299/check_mark.jpg" /> &#160;</td>
			<td>2270</td>
			<td>100-500</td>
			<td><img alt="Check Mark" src="/files/ftp_upload/54299/check_mark.jpg" /> &#160;</td>
			<td><img alt="Check Mark" src="/files/ftp_upload/54299/check_mark.jpg" /> &#160;&#160;</td>
		</tr>
		<tr>
			<td colspan="12">Tissue Sections</td>
		</tr>
		<tr>
			<td rowspan="2"></td>
			<td rowspan="2">Tissue Input&#160;</td>
			<td rowspan="2">Extracted Nucleic Acid</td>
			<td colspan="2">Validation</td>
			<td colspan="3">Quality check: DNA</td>
			<td colspan="4">Quality check: RNA</td>
		</tr>
		<tr>
			<td>(# of samples)</td>
			<td>Age of Sample (years)</td>
			<td>Total Yield (ng)</td>
			<td>Fragment size (bp)</td>
			<td>PCR</td>
			<td>Total Yield (ng)</td>
			<td>Fragment size (bp)</td>
			<td>PCR</td>
			<td>NanoString</td>
		</tr>
		<tr>
			<td>Heikal et al.&#160;</td>
			<td>5 x 5 &#181;m</td>
			<td>DNA</td>
			<td>12</td>
			<td>7-22</td>
			<td>88-300</td>
			<td>103-351</td>
			<td><img alt="Check Mark" src="/files/ftp_upload/54299/check_mark.jpg" /> &#160;&#160;</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chung et al.</td>
			<td>1 x 20 um</td>
			<td>RNA</td>
			<td>9</td>
			<td>&#62;5&#160;</td>
			<td></td>
			<td></td>
			<td></td>
			<td>16,000-&#160;&#160; 23,000</td>
			<td>100-200</td>
			<td><img alt="Check Mark" src="/files/ftp_upload/54299/check_mark.jpg" /> &#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Antica et al.&#160;</td>
			<td>2 x 4 &#181;m</td>
			<td>RNA</td>
			<td>18</td>
			<td>No data</td>
			<td></td>
			<td></td>
			<td></td>
			<td>Unknown (621 ng/&#181;l)</td>
			<td>80-202 +&#160;</td>
			<td><img alt="Check Mark" src="/files/ftp_upload/54299/check_mark.jpg" /> &#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ghatak et al.</td>
			<td>5 x 5 &#181;m</td>
			<td>DNA and RNA</td>
			<td>5</td>
			<td>1</td>
			<td>14 256</td>
			<td>&#60;1030&#160;</td>
			<td><img alt="Check Mark" src="/files/ftp_upload/54299/check_mark.jpg" /> &#160;</td>
			<td>16 000</td>
			<td>109-400 +</td>
			<td><img alt="Check Mark" src="/files/ftp_upload/54299/check_mark.jpg" /> &#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hennig et al.&#160;</td>
			<td>1 x 10 &#181;m&#160;</td>
			<td>DNA and RNA</td>
			<td>210</td>
			<td>1-25&#160;</td>
			<td>No data</td>
			<td>No data</td>
			<td><img alt="Check Mark" src="/files/ftp_upload/54299/check_mark.jpg" /> &#160;</td>
			<td>No data</td>
			<td>No data</td>
			<td><img alt="Check Mark" src="/files/ftp_upload/54299/check_mark.jpg" /> &#160;</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="12">Laser Capture Microdissection</td>
		</tr>
		<tr>
			<td rowspan="2"></td>
			<td rowspan="2">Tissue Input (mm2)</td>
			<td rowspan="2">Extracted Nucleic Acid</td>
			<td colspan="2">Validation</td>
			<td colspan="3">Quality check: DNA</td>
			<td colspan="4">Quality check: RNA</td>
		</tr>
		<tr>
			<td>(# of samples)</td>
			<td>Age of Sample (years)</td>
			<td>Total Yield (ng)</td>
			<td>Fragment size (bp)</td>
			<td>PCR</td>
			<td>Total Yield (ng)</td>
			<td>Fragment size (bp)</td>
			<td>PCR</td>
			<td>NanoString</td>
		</tr>
		<tr>
			<td>Snow et al.</td>
			<td>1-2</td>
			<td>DNA</td>
			<td>110</td>
			<td>0-2</td>
			<td>430</td>
			<td>No data</td>
			<td><img alt="Check Mark" src="/files/ftp_upload/54299/check_mark.jpg" /></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1:&#160; A comparison of published DNA and RNA extraction protocols for tissue cores, sections and laser capture microdissections. Also included are several molecular endpoints assessment of these methods using PCR and Nanostring assays.

Watch this Scientific Journal Video about Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction at JoVE.com

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

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 ...

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Research

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Biology

50.8K Views.  Queen's University.  Deze gewijzigde extractie protocol verbetert RNA en DNA opbrengst van meer doelgerichte regio's van belang in histopathologische weefsel blokken. 

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

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 ...

Extraction of Tissue Antigens for Functional Assays

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune diseases and cancer2. Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes 1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays 6. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.

A simple protocol for preparing extracts of human tissue to be used as a source of antigens in functional T-cell assays is described. This method allows T-cell responses to tissue-derived antigens to be measured in vitro.

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...

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 ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

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.

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 ...

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

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