The authors thank Ute Zeitz and Nikole Ginner for their excellent technical help as well as the Vetcore and animal care staff for their support.

Bone tissue from mice was used in strict accordance with prevailing guidelines for animal care and all efforts were made to minimize animal suffering.
1. Animals and Freeze Embedding
<ol>
	<li>House animals in conditions of constant room temperature (RT; 24 &#176;C) and a 12 h light/12 h dark cycle with free access to food and water. 
	NOTE: Bone tissues in this study were obtained from 3-month-old male wild type C57BL/6 mice.</li>
	<li>At necropsy, exsanguinate mice from the abdominal vena cava under general anesthesia (ketamine/xylazine, 100/6 mg/kg intraperitoneal). 
	NOTE: In order to avoid RNA degradation, use RNase-free instruments and materials, wear gloves and work quickly avoiding storage of samples at RT throughout the whole experiment.</li>
	<li>Remove whole femurs rapidly, clean them of surrounding soft tissues using scalpel and paper towels. Pour optimal cutting temperature (OCT) compound into the embedding mold, and put the femur into the bottom of the embedding mold. Snap-freeze the samples in liquid nitrogen. OCT is transparent at RT and white when frozen.</li>
	<li>Once entirely frozen, wrap the samples in foil and transfer them on dry ice to -80 &#176;C. Store samples at -80 &#176;C until further processing. 
	NOTE: The protocol can be paused here.</li>
</ol>
2. Section Preparation
<ol>
	<li>Set the temperature in the cryostat to -19 &#176;C and of the block holder to -17 &#176;C. Wipe the cryostat interior using 70% ethanol. Place the disposable blade for hard tissues, the glass slides and a fitting tool into the cryostat to cool. Keep them inside the cryostat for the duration of sectioning.</li>
	<li>Transfer the frozen tissue block on dry ice to the cryostat and allow it to equilibrate for at least 10 min. 
	NOTE: Avoid repetitive thawing and frequent cycling of one block from -80 &#176;C to -19 &#176;C for cryosectioning.</li>
	<li>Press the bottom of the embedding mold to push the OCT block out of the mold. Apply enough OCT medium to the block holder to adhere the block to it. Wait until the OCT medium is fully frozen.</li>
	<li>Place the block holder in the object holder and tighten it in place. Adjust the blade position and trim the block using 15 &#181;m cutting increments to remove the OCT covering the sample. If the sample has been cut earlier and the surface exposed to air, discard the first 2&#8722;3 tissue sections from the block.</li>
	<li>Adjust the cryostat to generate 8 &#181;m sections and cut 2&#8722;3 cryosections that will be discarded (the first few will typically be thicker than 8 &#181;m). Place the adhesive film on the block and use a fitting tool to adhere the film to the block. Make the cut slowly and at a constant speed holding the section by the film.</li>
	<li>Place the film (sample facing up) immediately on a precooled glass slide (on the cryobar within the cryostat) to avoid thawing of the sample. Use tape to fix the film to the glass slide for easier staining. Proceed immediately with the staining protocol.</li>
</ol>
3. Rapid Staining Protocol
<ol>
	<li>Prepare 40 mL of the following solutions in 50 mL tubes and put them on ice: 95% ethanol, RNase-free water, 100% ethanol, 100% ethanol and 100% xylene. Perform all steps on ice (except the staining). For each experimental day, prepare all aqueous reagents fresh with RNase-free water. 
	CAUTION: Work in the hood to avoid intoxication by xylene.</li>
	<li>Incubate sections for 30 s in 95% ethanol, then dip sections 30 s in RNase-free water to remove OCT carefully and completely which may interfere with LCM and downstream applications.</li>
	<li>Dispense 50 &#181;L of commercial LCM frozen section stain (Table of Materials) onto the section, incubate for 10 s at RT and drain it by placing the edge of the slide on absorbent tissue paper. Remove excess stain by rinsing 30 s in 100% ethanol.</li>
	<li>Immerse bone sections in a second tube with 100% ethanol for 30 s and transfer them to 100% xylene for 30 s.</li>
	<li>Put adhesive film (sample facing up) on dry glass slide as a support. Take care that the film is not impacted and placed as flat as possible. Do not allow the sample to dry completely.</li>
	<li>Place a PET membrane frame slide on it. Shortly press a gloved finger on the membrane to attach it to the film. The sample is then sandwiched between the membrane and the adhesive film. The adhesive film should not be folded or wrinkled and there should be no air bubbles between the film and the membrane. Proceed immediately with the LCM.</li>
</ol>
4. Laser Capture Microdissection
<ol>
	<li>Clean the stage and cap holder from RNase using surface decontaminant (Table of Materials). Load the slide and the caps in the slide holder and the cap holder, respectively.</li>
	<li>Adjust the focus and acquire slide overview with 1.25x objective. Change to the 40x objective and adjust the focus. Choose the area of interest using the slide overview. Adjust laser parameters as follows: aperture, 7; laser power, 60; speed, 5; pulse frequency, 201; specimen balance, 20. Optimize these parameters for each objective.</li>
	<li>If the laser fails to cut the sample, increase the laser power. Alternatively, the laser can be applied more than once. In addition, inspect the target for spots of incomplete cuts and re-drawn the line in these spots using the Move and cut option. Save laser settings for the dissection of subsequent slides.</li>
	<li>Select osteoblasts, osteocytes and bone lining cells in distal femoral cancellous or cortical bone based on morphologic criteria. Draw a line for laser path further away from the target cells to minimize the damage by the UV laser. Perform all microdissections within less than 1 h after the staining.</li>
	<li>Collect each cell type in a separate cap of a 0.5 mL tube. 
	NOTE: Dry capturing instead of liquid recovery may avoid RNA degradation. In addition, very small volumes of buffer contained in the cap of the microcentrifuge tube can either evaporate or crystallize (depending on its composition) during the LCM.</li>
	<li>Confirm capture success by observation of the collection tube cap after the LCM where applicable. Proceed immediately with the RNA extraction.</li>
</ol>
5. RNA Extraction
<ol>
	<li>Dispense 50 &#181;L of the lysis buffer containing &#946;-mercaptoethanol into the cap of the collection tube and lyse the sample by pipetting up and down in the cap for 1 min. Spin down the lysate and add 300 &#181;L of the lysis buffer containing &#946;-mercaptoethanol into the tube. If several caps are going to be pooled, take care that the total volume of buffer is as recommended for the RNA extraction kit (Table of Materials). 
	CAUTION: &#946;-mercaptoethanol must be added to protect RNA from degradation, but it is considered toxic. Wear protective clothes and gloves and work in the hood.</li>
	<li>In addition, for each slide prepare one labeled 1.5 mL microcentrifuge tube with 350 &#181;L of the lysis buffer containing &#946;-mercaptoethanol. Use the sections remaining after the LCM to extract RNA. Carefully separate the film from the membrane and lyse the sample by slowly pipetting the lysis buffer onto the section several times. 
	NOTE: Total amount of lysis buffer should be 350 &#181;L. Do not use all of it at once for digestion as it will flow off the slide.</li>
	<li>Put the lysate samples on dry ice and store them at -80 &#176;C. 
	NOTE: The protocol can be paused here.</li>
	<li>Thaw the lysates at RT. Transfer lysates from LCM-harvested cells from collection tubes into the 1.5 mL microcentrifuge tubes. If more than one cap was used to harvest the sample, pool several lysates.</li>
	<li>Extract RNA according to the manufacturer&#8217;s instructions. Treat columns with DNase I to remove genomic DNA. Elute RNA with 14 &#181;L of RNase-free water, resulting in a 12 &#181;L eluate. Store RNA at -80 &#176;C. 
	NOTE: The protocol can be paused here.</li>
</ol>
6. Measurement of RNA Yield and Integrity
<ol>
	<li>Thaw RNA on wet ice. Measure the yield and integrity of isolated RNA using micro-capillary electrophoresis. Load total RNA (1 &#181;L per sample) into a chip (Table of Materials) according to the manufacturer&#8217;s instructions.</li>
</ol>

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The authors have nothing to disclose.

Both RNA quality and quantity can be affected negatively at all stages of the sample preparation such as tissue manipulation, LCM process, and RNA extraction. Therefore, an LCM protocol was developed to obtain sufficient amount of high-quality RNA for subsequent gene expression analysis.
For LCM, most laboratories use sections 7&#8722;8 &#181;m thick2. Thicker sections would allow more material to be harvested. However, if they are too thick, this could reduce the microscopic resolution and the laser may not be able to cut through. It is likely that optimal tissue thickness depends on the LCM system (and the laser and slide type) used, as well as on the tissue in question8,25. In the present study, cryosections of different thicknesses (4, 8 or 12 &#181;m) were tested; 8 &#181;m-thick sections were found to be ideal for LCM, while 12 &#181;m bone sections were more difficult to cut with the laser and 4 &#181;m sections gave lower amounts of RNA.
The endogenous RNase activity varies between different tissues. Therefore, staining protocols developed for tissues with very low RNase activity could be unsuitable for other tissue types. Nuclear fast red26 and cresyl violet24 were suggested to be the best in terms of preserving RNA integrity. In addition, alcohol-based methods were superior to aqueous stains for maintaining RNA integrity. However, they suffered from irreproducible staining intensity27. Time is an important parameter to consider. On the one hand, the time required to search and identify cells of interest in the section, and to perform LCM is often relatively long. On the other hand, time available for LCM is limited, since, in some cases, 20% RNA degradation was reached after 30 min28. Therefore, different methods were applied in order to stabilize RNA, such as exposure of sections to argon flux during the microdissection28, or use of buffered alcohol-based cresyl violet staining and keeping the humidity level in the laboratory low27. In addition, a maximum of 15 min for the microdissection step was suggested2. In this study, frozen bone sections were stained using a rapid protocol for a commercial LCM stain, and even after 1 h at RT, RIN values decreased from 8.3 to 9.1. Therefore, all microdissections were performed within less than 1 h after staining. In addition, different protocols for staining were tested (with shorter incubations in aqueous reagents or without the xylene step). No significant improvement in RIN values was achieved.
In LCM studies, two different types of RNA extraction methods have been compared: a phase separation method vs. a column-based method. It was found that the RNA extraction method based on columns led to better RNA quality and higher yield compared to the phase separation method8. In another study, a commercial kit that uses minimal digestion time and temperature (5 min at RT) resulted in superior RNA quality compared to an RNA isolation kit that needs longer digestion time at higher temperature (30 min at 42 &#176;C)7. In the present study, it was possible to extract high quality RNA (RIN &#62; 8) of sufficient concentration from LCM samples using fresh-frozen mouse bones and a column-based RNA extraction method. Thorough lysis (1 min pipetting in the cap) is essential for good RNA yield. The effect of the RNA extraction method on the RNA yield and integrity obtained after LCM was investigated using several different RNA isolation kits according to the manufacturers&#8217; instructions. However, better quality RNAs and increased quantity were obtained with the kit used in the optimized protocol. In the pilot study, microdissected tissue regions of 1 mm2 final area were cut, and it was possible to isolate approximately 8.5 ng of RNAs using 8-&#181;m-thick bone sections (approximately 800 pg/&#181;L). Typically, osteoblasts, osteocytes, and bone lining cells were captured in 2&#8722;3 sections per sample.
Direct comparisons between different LCM instruments are scarce. In one study, two common LCM instruments were tested, which differ in the type of laser used (UV and infrared [IR], respectively) and method of capturing tissue (adhesive isolation cap vs. caps coated with transparent thermoplastic film, respectively). It was found that for thinly-sectioned fresh-frozen mouse brain sections, the IR system resulted in modestly higher RNA quality7. In the present study, two LCM systems that allow contact-free sample collection were compared. In one system, the microdissected sample falls by gravity into the cap placed just below29. Another system uses a de-focused laser pulse, which catapults the material into the overhanging cap30. For fresh frozen bones, higher RNA amounts and RIN values were obtained when gravity was used for tissue capturing. In addition, the catapulting technology is not compatible with the &#8220;sandwich&#8221; composed of adhesive film, cryo-section, and PET membrane, due to the additional weight of the PET membrane.
LCM requires physical access to the tissue surface. Therefore, mounting and glass covering of the specimen is inapplicable, with the consequence of impaired visualization of morphology. To overcome this limitation, a fluid cover medium was developed31. Alternatively, 10&#8722;15 &#181;L of ethanol can be added directly on the tissue section, enabling better morphological analysis before evaporation2. In the present study, frozen bone sections were cut on an adhesive film and, before the sample was allowed to dry completely, it was sandwiched between the PET membrane and the film. This &#8220;sandwich&#8221; method gave good morphological contrast and specific bone cells were clearly identified.

Tissues are made up of heterogeneous and spatially distributed cell types. Different cell types in a given tissue may respond differently to the same signal. Therefore, it is essential being able to isolate specific cell populations for the assessment of the role of different cell types in both physiological and pathological conditions. Laser capture microdissection (LCM) offers a relatively rapid and precise method for isolating and removing specified cells from complex tissues1. LCM systems use the power of a laser beam to separate the cells of interest from histological tissue sections without the need for enzymatic processing or growth in culture. This means that the cells are in their natural tissue habitat, and that tissue architecture including the spatial relationship between different cells is retained. Morphology of both the captured cells and the residual tissue is well preserved, and several tissue components can be sampled sequentially from the same slide. Isolated cells can then be used for subsequent analysis of their RNA, DNA, protein or metabolite content2,3.
In order to analyze gene expression in different cell populations, or after different treatments, it is necessary to obtain mRNAs of sufficient quality and quantity for the subsequent analysis4,5. In contrast to DNA, RNAs are more sensitive to fixation and the use of frozen tissue is recommended when the objective is to study RNA. Since mRNAs are quickly degraded by ubiquitous ribonucleases (RNase), stringent RNase-free conditions during specimen handling and preparation and avoiding storage of samples at room temperature are required. In addition, rapid techniques without any prolonged aqueous phase steps are crucial to prevent RNA degradation6. The RNA yield and integrity can also be affected by the LCM process and the LCM system used7,8. Currently, four LCM systems with different operating principles are available2. The method of RNA extraction can also be important, since different RNA isolation kits have been tested with significant differences in RNA quantity and quality7,8.
Any tissue preparation method requires finding a balance between obtaining good morphological contrast and maintaining RNA integrity for further analyses. For preparing frozen sections from bone, an adhesive film was developed and continuously improved9. The bone sections are cut and stained directly on the adhesive film. This adhesive film is applicable to many types of staining, and can be employed to isolate the cells of interest from bone cryosections using LCM9,10,11,12,13,14. All the steps including the surgical removal, embedding, freezing, cutting and staining can be completed within less than one hour. Importantly, cells such as osteoblasts, bone lining cells, and osteoclasts can be clearly identified9,10,11,12,13,14. This method has the advantage of being rapid and simple. An alternative method for generating bone cryosections is to use the tape transfer system15. However, the latter technique is more time-consuming and requires additional instrumentation, since the sections have to be transferred from the adhesive tape onto precoated membrane slides by ultraviolet (UV) cross-linking. Although the tape transfer system has been successfully coupled with LCM16,17,18,19, it should be noted that the cross-linked coating can create a background pattern that can interfere with cell-type identification20.
Typically, only small amounts of RNA are extracted from microdissected cells, and RNA quality and quantity are often assessed by micro-capillary electrophoresis21. A computer program is used to assign an index of quality to RNA extracts called RNA integrity number (RIN). A RIN value of 1.0 indicates completely degraded RNA, whereas a value of 10.0 suggests that the RNA is fully intact22. Usually, indexes over 5 are considered sufficient for RNA studies. Gene expression patterns in samples with an RIN value of 5.0&#8722;10.0 have been reported to correlate well with each other23. Although the sensitivity of this method is high, since as little as 50 pg/&#181;L of total RNA can be detected, it can be very difficult to obtain a quality assessment if the RNA concentration in the sample is very low. Therefore, in order to assess RNA quality, the tissue section remaining after the LCM is often used to extract RNA, by pipetting buffer onto the slide24.
Although LCM has been used extensively on different frozen tissues, RIN values of extracted RNAs are rarely reported. Furthermore, there are no comparative studies to clarify the most appropriate method to study RNA in mouse bones. In the present study, frozen sections from adult mouse femurs were used to optimize sample preparation, LCM protocol and RNA extraction in order to obtain high quality RNAs. The present protocol was optimized particularly for the LCM system that uses gravity for sample collection.

<table>
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>63689-25ML-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethanol EMPLURA</td>
			<td>Merck Millipore</td>
			<td>8,18,76,01,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive film (LMD film)</td>
			<td>Section-Lab</td>
			<td>C-FL001</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer System</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent RNA 6000 Pico Chip Kit</td>
			<td>Agilent Technologies</td>
			<td>5067-1513</td>
			<td></td>
		</tr>
		<tr>
			<td>Arcturus HistoGene Staining Solution</td>
			<td>Applied Biosystems</td>
			<td>12241-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryofilm fitting tool</td>
			<td>Section-Lab</td>
			<td>C-FT000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat Leica CM 1950</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glass microscope slides, cut colour frosted orange</td>
			<td>VWR Life Science</td>
			<td>631-1559</td>
			<td></td>
		</tr>
		<tr>
			<td>Histology tissue molds PVC</td>
			<td>MEDITE</td>
			<td>48-6302-00</td>
			<td></td>
		</tr>
		<tr>
			<td>LMD7&#160;Laser&#160;Mikrodissektion&#160;System</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low profile Microtome Blades Leica DB80 XL</td>
			<td>Leica Biosystems</td>
			<td>14035843496</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>VWR Life Science</td>
			<td>E476-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>PET membrane slides 1.4 mircon</td>
			<td>Molecular Machines &#38; Industries GmbH</td>
			<td>50102</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Away surface decontaminant</td>
			<td>Molecular BioProduct</td>
			<td>7002</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Micro Kit</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek optimal cutting temperature (OCT) compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR Life Science</td>
			<td>2,89,73,363</td>
			<td></td>
		</tr>
	</tbody>
</table>

a laser capture microdissection protocol that yields high quality rna from fresh-frozen mouse bones

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-&#181;m-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

An LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells of mouse femurs. In the optimized protocol, 8 &#181;m-thick frozen bone sections were cut on an adhesive film and stained using a rapid protocol for a commercial LCM frozen section stain. The sample was sandwiched between the PET membrane and the adhesive film. Mouse bone cells were microdissected using an LCM system that uses gravity for sample collection. A column-based RNA extraction method was used to obtain a high-quality RNA of sufficient yield. The yield and integrity of isolated RNA was measured using micro-capillary electrophoresis (Figure 1).
The difference in RNA quality and quantity obtained using different lysis protocols can be seen in the representative gel and electropherograms. When the sample was lysed by pipetting up and down in the cap for 1 min, it was possible to isolate approximately 8.5 ng of RNA from 1 mm2 microdissected bone tissue (8 &#181;m-thick section). RIN value was 8.60 (Figure 2).
Alternatively, LCM was performed using an LCM system that uses de-focused laser pulse, which catapults the material into the overhanging adhesive cap. RNA quality and quantity can be estimated in the representative gel and electropherograms. For fresh frozen bones, it was possible to isolate approximately 1.6 ng of RNA from 1 mm2 microdissected bone tissue (8 &#181;m-thick section). The RIN value was 1 (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60197/60197fig01.jpg" /> 
Figure 1: Flowchart of laser capture microdissection protocol for fresh-frozen bones. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60197/60197fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60197/60197fig02.jpg" /> 
Figure 2: Representative gel (top) and electropherograms (bottom) of RNA samples.&#160;LCM was performed using an LCM system that uses gravity for sample collection. RNA samples were retrieved from LCM-harvested tissue (samples 1, 3, 5, 7, and 9) and corresponding control sections (samples 2, 4, 6, 8, and 10, respectively). One RNA sample was retrieved from the control section that was stained but was not used for LCM (sample 11). A total area of 1 mm2 was microdissected, and different lysis protocols were used. Sample 1: 350 &#181;L of the lysis buffer containing &#946;-mercaptoethanol were added into the tube. The cap was closed carefully, and the tube inverted. LCM-harvested cells were lysed by vortexing 1 min, incubating at RT for 10 min and vortexing 1 min. Sample 3: 350 &#181;L of the lysis buffer containing &#946;-mercaptoethanol was added into the collection tube, the cap was carefully closed, and the tube inverted. LCM-harvested cells were lysed by incubating collection tubes upside down for 30 min at RT. Sample 5: 50 &#181;L of the lysis buffer containing &#946;-mercaptoethanol was added into the cap of the collection tube and the sample was lysed by pipetting up and down in the cap for 1 min. The lysate was centrifuged and 300 &#181;L of the lysis buffer containing &#946;-mercaptoethanol was added into the tube. Sample 7: 350 &#181;L of the lysis buffer containing &#946;-mercaptoethanol was added into the tube. The cap was closed carefully, and the tube inverted. LCM-harvested cells were lysed by vortexing and inverting several times. Sample 9: 50 &#181;L of the lysis buffer containing &#946;-mercaptoethanol was added into the cap of the collection tube, and the sample was lysed by incubation for 5 min at RT in the cap. The lysate was centrifuged, and 300 &#181;L of the lysis buffer containing &#946;-mercaptoethanol was added to the tube. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60197/60197fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60197/60197fig03.jpg" /> 
Figure 3: Representative gel (top) and electropherograms (bottom) of RNA samples.&#160;LCM was performed using an LCM system that uses de-focused laser pulse, which catapults the material into the adhesive cap positioned above the section. RNA samples were retrieved from LCM-harvested tissue (samples 5 and 6) and corresponding control section (sample 7). One RNA sample was retrieved from the control section that was stained but was not used for LCM (sample 8). A total area of 0.5 mm2 or 1 mm2 was microdissected (samples 5 and 6, respectively). 350 &#181;L of the lysis buffer containing &#946;-mercaptoethanol was added into the collection tube, the cap was carefully closed and the tube inverted. LCM-harvested cells were lysed by incubating collection tubes upside down for 30 min at RT. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60197/60197fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones at JoVE.com

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser ...

a-laser-capture-microdissection-protocol-that-yields-high-quality-rna

Nanyang Technological University

Research

JoVE Journal

Biology

9.4K Views.  University of Veterinary Medicine Vienna. A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

Video: A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...

A Web Tool for Generating High Quality Machine-readable Biological Pathways

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological information. The pathways generated by this online application are machine-readable and fully compatible with essentially all web-browsers and computer operating systems. It uses a specially developed, web-enabled pathway drawing interface that permits the selection and placement of different combinations of pre-drawn biological or biochemical entities to depict reactions, interactions, transport processes and binding events. This palette of entities consists of chemical compounds, proteins, nucleic acids, cellular membranes, subcellular structures, tissues, and organs. All of the visual elements in it can be interactively adjusted and customized. Furthermore, because this tool is a web server, all pathways and pathway elements are publicly accessible. This kind of pathway &#34;crowd sourcing&#34; means that PathWhiz already contains a large and rapidly growing collection of previously drawn pathways and pathway elements. Here we describe a protocol for the quick and easy creation of new pathways and the alteration of existing pathways. To further facilitate pathway editing and creation, the tool contains replication and propagation functions. The replication function allows existing pathways to be used as templates to create or edit new pathways. The propagation function allows one to take an existing pathway and automatically propagate it across different species. Pathways created with this tool can be &#34;re-styled&#34; into different formats (KEGG-like or text-book like), colored with different backgrounds, exported to BioPAX, SBGN-ML, SBML, or PWML data exchange formats, and downloaded as PNG or SVG images. The pathways can easily be incorporated into online databases, integrated into presentations, posters or publications, or used exclusively for online visualization and exploration. This protocol has been successfully applied to generate over 2,000 pathway diagrams, which are now found in many online databases including HMDB, DrugBank, SMPDB, and ECMDB.

PathWhiz is a comprehensive, online pathway drawing tool for generating biochemical and biological pathways. It uses publicly accessible databases and easily expandable palettes consisting of pre-drawn pathway components. This protocol describes how to easily build new pathways, replicate and edit existing pathways, and propagate previously drawn pathways to different organisms.

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma. However, the precise molecular mechanisms involved are still poorly understood. The ability to perform gene expression analysis will be crucial in obtaining further insights into the function of these cells and its role in the regulation of IOP and glaucoma development. To achieve this, a reproducible method for isolating highly enriched TM from frozen sections of mouse eyes and a method for downstream gene expression analysis, such as RT-qPCR and RNA-Seq is needed. The method described herein is developed to isolate highly pure TM from mouse eyes for downstream digital PCR and microarray analysis. In addition, this technique can be easily adapted for the isolation of other highly enriched ocular cells and cell compartments that have been difficult to isolate from mouse eyes. The combination of LCM and RNA analysis can contribute to a more comprehensive understanding of the cellular events underlying glaucoma.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

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