Name: a novel microdissection approach to recovering mycobacterium tuberculosis specific transcripts from formalin fixed paraffin embedded lung granulomas
Uploaded: 2014-06-05T14:00:00
Description: Watch this Scientific Journal Video about A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas at JoVE.c

The authors would like to acknowledge the following NIH awards/subawards for the support of this research: R01HL106790, R01HL106790-S1, R01HL106786, R01AI089323, R21AI091457, R21RR026006, P20RR020159, C06RR017563, 8T32OD011124-08, and P51OD011104.

1. Calibrate Mesodissection Instrument with 2iD Imaging Software
The 2iD imaging software will be referred to as either the software or the program for the remainder of this article. This step is needed to correctly track the image as well as align multiple slide frames.
<ol>
	<li>Turn on instrument and computer.</li>
	<li>Open software and select &#8220;calibrate instrument&#8221;.</li>
	<li>Calibrate stage travel by using the joystick. Move stage to upper left position. Press &#8220;set&#8...

<ol>
	<li>Fend, F., Raffeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+in+pathology.">Laser capture microdissection in pathology.</a> J Clin Pathol. 53 (9), 666-672 (2000).</li><li>Esposito, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementary+techniques:+laser+capture+microdissection--increasing+specificity+of+gene+expression+profiling+of+cancer+specimens.">Complementary techniques: laser capture microdissection--increasing specificity of gene expression profiling of cancer specimens.</a> Adv Exp Med Biol. 593, 54-65 (2007).</li><li>Adey, N., Bosh, D., Emery, D., Birch, L., Parry, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesodissection+of+Paraffin+Embedded+Slide+Mounted+Tissue+Sections.">Mesodissection of Paraffin Embedded Slide Mounted Tissue Sections.</a> Journal of Molecular Diagnostics. 14 (6), (2012).</li><li>Fleischmann, R. 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Mesodissection is a technique that can be applied to RNA extraction from pathologic lesion slides caused by a vast array of pathogens. It is a necessity that the user tracks the image and aligns the image correctly. To accomplish this, the instrument must be calibrated following the directions on the imaging software. When dissecting, if the slide and area of interest being dissected do not align, the user must recalibrate the instrument. Another critical step in the dissection process is to load the consumable mill bit ...

Samples have traditionally been manually microdissected from either whole tissue or slides using a needle and scalpel. This necessitates a clear separation between the tissue section of interest and surrounding tissues sections2. With the current advancements in molecular profiling technology, there has been an increasing need to assess tissue at the cellular level. Due to the limitations of manual microdissection, techniques including LCM were established to allow greater isolation precision. This technique allows the researcher to isolate specific cell populations from various cell and slide types, which can then be used for downstream assays such as gene expression profiling. Although very effective for downstream assays, LCM is not without limitations. First, LCM is an expensive and time consuming process. Additionally, due to the unstable nature of RNA, it is often challenging to obtain high quality RNA from LCM samples2. Due to the disadvantages of LCM, new advances in microdissection technology are still needed to make it more accessible to a greater number of researchers in an affordable and time sensitive manner.
One such advancement in microdissection technology now available is a technique known as mesodissection. In this technique a machine is used to mill the annotated tissue section of interest and aspirates it into a consumable mill bit3. Next, this sample can be aspirated into a collection tube and used for downstream applications. The advantages of this system are that it is significantly less expensive and time sensitive. In our experience, the system allows the dissection of a lung granuloma tissue section in ten minutes. On the other hand, traditional LCM would likely require hours to complete the process. The system allows the operator to load a reference slide to use as a comparison as well as tool for annotation. Additionally a report is generated outlining the area of the slide that was dissected. There are two main disadvantages to mesodissection. Although effective at extracting multiple cells, it is challenging to isolate a single cell. In addition, the precision of the image generated is not as clear as if using other imaging microscopes.
Tuberculosis (TB) is a major infectious disease killer of humanity worldwide and results from infection with Mtb. In a majority of individuals exposed to aerosols of Mtb, the infection is latently limited. In at least 10 million people annually, it results in active TB disease4. During latent infection, Mtb is contained within pathological lung lesions known as granulomas. Hence it has been argued that the outcome of Mtb infection is decided at the level of the granuloma5.
Here we demonstrate how mesodissection can be used to microdissect granulomas caused by Mtb. The slides used are from FFPE lung tissue from infected rhesus macaques. For the purpose of this demonstration, we will dissect granulomas in their entirety. We also demonstrate that RNA can be extracted from the recovered tissue. This technique can be applied to tissue samples from various other specimens and then used for a variety of downstream assays.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:260px;">MeSectr</td>
			<td style="width:113px;">AvanSci Bio</td>
			<td style="width:108px;"></td>
			<td style="width:195px;">Mesodissector</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">400 nm xScisors</td>
			<td>AvanSci Bio</td>
			<td></td>
			<td>Other sizes available</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">THOR</td>
			<td>AvanSci Bio</td>
			<td></td>
			<td>Programmable Heater-Shaker</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NanoDrop2000</td>
			<td>ThermoScientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy FFPE extraction kit&#160;</td>
			<td>Qiagen</td>
			<td align="right" style="width:108px;">73504</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ovation RNA-Seq FFPE System&#160;</td>
			<td>Nugen</td>
			<td align="right">7150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick PCR Purification Kit&#160;</td>
			<td>Qiagen</td>
			<td align="right" style="width:108px;">28104</td>
			<td style="width:195px;"></td>
		</tr>
	</tbody>
</table>

a novel microdissection approach to recovering mycobacterium tuberculosis specific transcripts from formalin fixed paraffin embedded lung granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

The aforementioned protocol shows how to use a new mesodissection technique to extract RNA from FFPE tissue slides. This protocol&#8217;s efficacy is shown through FFPE slides of lung granulomas from NHP infected with Mtb in various infective stages. Figures 1-3&#160;are images of the instrument. Figure 4 depicts how the dissection process occurs and the resulting image generated by the software. Table 1 shows the results of RNA extraction with a granuloma from ...

Watch this Scientific Journal Video about A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas at JoVE.c

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

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