Name: a laser capture microdissection protocol that yields high quality rna from fresh-frozen mouse bones
Uploaded: 2019-09-16T15:00:00
Description: Watch this Scientific Journal Video about A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones at JoVE.com

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 fr...

<ol>
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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphiregulin+lacks+an+essential+role+for+the+bone+anabolic+action+of+parathyroid+hormone.">Amphiregulin lacks an essential role for the bone anabolic action of parathyroid hormone.</a> Molecular and Cellular Endocrinology. 417, 158-165 (2015).</li><li>Murali, S. K., Andrukhova, O., Clinkenbeard, E. L., White, K. E., Erben, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+Osteocytic+Fgf23+Secretion+Contributes+to+Pyrophosphate+Accumulation+and+Mineralization+Defect+in+Hyp+Mice.">Excessive Osteocytic Fgf23 Secretion Contributes to Pyrophosphate Accumulation and Mineralization Defect in Hyp Mice.</a> PLoS Biology. 14 (4), e1002427 (2016).</li><li>Andrukhova, O., Sch&#252;ler, C., Bergow, C., Petric, A., Erben, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Augmented+Fibroblast+Growth+Factor-23+Secretion+in+Bone+Locally+Contributes+to+Impaired+Bone+Mineralization+in+Chronic+Kidney+Disease+in+Mice.">Augmented Fibroblast Growth Factor-23 Secretion in Bone Locally Contributes to Impaired Bone Mineralization in Chronic Kidney Disease in Mice.</a> Frontiers in Endocrinology. 9, 311 (2018).</li><li>Golubeva, Y. G., Smith, R. M., Sternberg, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+Frozen+Sample+Preparation+for+Laser+Microdissection:+Assessment+of+CryoJane+Tape-Transfer+System&#174;.">Optimizing Frozen Sample Preparation for Laser Microdissection: Assessment of CryoJane Tape-Transfer System&#174;.</a> PLoS ONE. 8 (6), e66854 (2013).</li><li>Pacheco, E., Hu, R., Taylor, S. <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+and+Transcriptional+Analysis+of+Sub-Populations+of+the+Osteoblast+Lineage+from+Undecalcified+Bone.">Laser Capture Microdissection and Transcriptional Analysis of Sub-Populations of the Osteoblast Lineage from Undecalcified Bone.</a> Methods in Molecular Biology (Clifton, N.J.). 1723, 191-202 (2018).</li><li>Nioi, P., 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=Transcriptional+Profiling+of+Laser+Capture+Microdissected+Subpopulations+of+the+Osteoblast+Lineage+Provides+Insight+Into+the+Early+Response+to+Sclerostin+Antibody+in+Rats.">Transcriptional Profiling of Laser Capture Microdissected Subpopulations of the Osteoblast Lineage Provides Insight Into the Early Response to Sclerostin Antibody in Rats.</a> Journal of Bone and Mineral Research. 30 (8), 1457-1467 (2015).</li><li>Taylor, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+time-dependent+transcriptional+changes+in+the+osteoblast+lineage+in+cortical+bone+associated+with+sclerostin+antibody+treatment+in+ovariectomized+rats.">Differential time-dependent transcriptional changes in the osteoblast lineage in cortical bone associated with sclerostin antibody treatment in ovariectomized rats.</a> Bone Reports. 8, 95-103 (2018).</li><li>Taylor, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-dependent+cellular+and+transcriptional+changes+in+the+osteoblast+lineage+associated+with+sclerostin+antibody+treatment+in+ovariectomized+rats.">Time-dependent cellular and transcriptional changes in the osteoblast lineage associated with sclerostin antibody treatment in ovariectomized rats.</a> Bone. 84, 148-159 (2016).</li><li>Martin, L. B. B., 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=Laser+microdissection+of+tomato+fruit+cell+and+tissue+types+for+transcriptome+profiling.">Laser microdissection of tomato fruit cell and tissue types for transcriptome profiling.</a> Nature Protocols. 11 (12), 2376-2388 (2016).</li><li>Imbeaud, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+standardization+of+RNA+quality+assessment+using+user-independent+classifiers+of+microcapillary+electrophoresis+traces.">Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces.</a> Nucleic Acids Research. 33 (6), e56 (2005).</li><li>Schroeder, 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=The+RIN:+an+RNA+integrity+number+for+assigning+integrity+values+to+RNA+measurements.">The RIN: an RNA integrity number for assigning integrity values to RNA measurements.</a> BMC Molecular Biology. 7, 3 (2006).</li><li>Gallego Romero, I., Pai, A. A., Tung, J., Gilad, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-seq:+impact+of+RNA+degradation+on+transcript+quantification.">RNA-seq: impact of RNA degradation on transcript quantification.</a> BMC Biology. 12, 42 (2014).</li><li>Bevilacqua, C., Makhzami, S., Helbling, J. C., Defrenaix, P., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintaining+RNA+integrity+in+a+homogeneous+population+of+mammary+epithelial+cells+isolated+by+Laser+Capture+Microdissection.">Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection.</a> BMC Cell Biology. 11, 95 (2010).</li><li>Mahalingam, M., Murray, G. I. <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:+Insights+into+Methods+and+Applications.">Laser Capture Microdissection: Insights into Methods and Applications.</a> Laser Capture Microdissection: Methods and Protocols. , 1-17 (2018).</li><li>Burgemeister, R., Gangnus, R., Haar, B., Sch&#252;tze, K., Sauer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+quality+RNA+retrieved+from+samples+obtained+by+using+LMPC+(laser+microdissection+and+pressure+catapulting)+technology.">High quality RNA retrieved from samples obtained by using LMPC (laser microdissection and pressure catapulting) technology.</a> Pathology, Research and Practice. 199 (6), 431-436 (2003).</li><li>Cummings, 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=A+robust+RNA+integrity-preserving+staining+protocol+for+laser+capture+microdissection+of+endometrial+cancer+tissue.">A robust RNA integrity-preserving staining protocol for laser capture microdissection of endometrial cancer tissue.</a> Analytical Biochemistry. 416 (1), 123-125 (2011).</li><li>Cl&#233;ment-Ziza, M., Munnich, A., Lyonnet, S., Jaubert, F., Besmond, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilization+of+RNA+during+laser+capture+microdissection+by+performing+experiments+under+argon+atmosphere+or+using+ethanol+as+a+solvent+in+staining+solutions.">Stabilization of RNA during laser capture microdissection by performing experiments under argon atmosphere or using ethanol as a solvent in staining solutions.</a> RNA. 14 (12), 2698-2704 (2008).</li><li>K&#246;lble, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+LEICA+microdissection+system:+design+and+applications.">The LEICA microdissection system: design and applications.</a> Journal of Molecular Medicine. 78 (7), B24-B25 (2000).</li><li>B&#246;hm, M., Wieland, I., Sch&#252;tze, K., R&#252;bben, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbeam+MOMeNT:+non-contact+laser+microdissection+of+membrane-mounted+native+tissue.">Microbeam MOMeNT: non-contact laser microdissection of membrane-mounted native tissue.</a> The American Journal of Pathology. 151 (1), 63-67 (1997).</li><li>Micke, P., 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=A+fluid+cover+medium+provides+superior+morphology+and+preserves+RNA+integrity+in+tissue+sections+for+laser+microdissection+and+pressure+catapulting.">A fluid cover medium provides superior morphology and preserves RNA integrity in tissue sections for laser microdissection and pressure catapulting.</a> The Journal of Pathology. 202 (1), 130-138 (2004).</li></ol>

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 resolu...

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 us...

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 ...

Have you ever thought about harnessing the power of laser capture microdissection for analysis of gene expression in specific bone cells within their natural environment? Here, we describe a protocol that will enable you to obtain sufficient quantities of high quality RNA from cryosections of mouse bones. This technology is a great tool for examining changes in gene expression in specific bone cells in genetically engineered mouse models or in disease models.The protocol described here is focused on mouse bones, but can be used to study in situ a gene expression in cells of any hard tissue in any species. Helping to demonstrate this procedure, will be Christiane Schueler, a technician from my laboratory. After euthanizing mice by exsanguination under general anesthesia, remove whole femurs rapidly.Use a scalpel and paper towels to clean the femurs of the surrounding soft tissues. Next, pour optimal cutting temperature compound into the embedding mold and place the femurs into the bottom of the embedding molds. Snap freeze the samples in liquid nitrogen.Once the samples are entirely frozen, wrap them in foil and transfer them on dry ice to a freezer. Store them at minus 80 degrees Celsius until ready for further processing. First, set the temperature in the cryostat to minus 19 degrees Celsius and the temperature of the block holder to minus 17 degrees Celsius.Next, wipe down the interior of the cryostat with 70%ethanol. Place the disposable blade for hard tissues, the glass slides, and a fitting tool into the cryostat to cool, and keep them inside the cryostat for the duration of sectioning. Transfer the frozen tissue block on dry ice to the cryostat and allow it to equilibrate for at least 10 minutes.Then, 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 and wait until the OCT medium freezes completely. After this, place the block holder in the object holder and tighten it in place.Adjust the blade position and trim the block in 15-micrometer cutting increments to remove the OCT covering the sample. Adjust the cryostat to generate eight-micrometer sections and cut two to three cryosections that will be discarded. Place adhesive film on the block and use the fitting tool to adhere the film to the block.Now, make a cut slowly and at constant speed, while holding the section by the film. Place the film with the sample facing up on a pre-cooled 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.In a fume hood, prepare the necessary solutions of ethanol, RNase-free water, and xylene on ice as outlined in the text protocol. Incubate the sections in 95%ethanol for 30 seconds. Then, carefully dip the sections in RNase-free water for 30 seconds to completely remove the OCT.Next, dispense 50 microliters of commercial LCM frozen section stain onto the section and incubate at room temperature for 10 seconds. Drain the section by placing the edge of the slide on absorbent tissue paper. Rinse the section in 100%ethanol for 30 seconds to remove any excess stain.Immerse the bone sections in a second tube containing 100%ethanol for 30 seconds and then, transfer them to 100%xylene for 30 seconds. Put adhesive film on a dry glass slide as a support, taking care to place the film as flat as possible. After this, place a PET membrane frame slide on the film and briefly press a gloved finger on the membrane to attach it to the film.This sample will be 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. Start by using surface decontaminant to clean the stage end cap holder.Load the slide into the slide holder and the caps into the cap holder. Next, adjust the focus and acquire a slide overview with the 1.25x objective. Change to the 40x objective and adjust the focus.Using the slide overview, choose the area of interest. Then, adjust the laser parameters as shown here, making sure to optimize these parameters for each objective. If the laser fails to cut the sample, increase the laser power.Select osteoblast, osteocites, and bone lining cells in distal femoral cancellous or cortical bone based on morphologic criteria. Draw a line for the laser path further away from the target cells to minimize the damage by the UV laser. If the laser fails to cut the sample, apply the laser more than once or inspect the target for spots of incomplete cuts and use the move and cut option to cut the tissue in these spots.Collect each cell type in a separate 0.5-milliliter tube cap. First, dispense 50 microliters of the lysis buffer containing beta mercaptoethanol into the cap of the collection tube. Lyse the sample by pippetting it up and down in the cap for one minute.Next, spin down the lysate and add 00 microliters of the lysis buffer containing beta-mercaptoethanol to the tube. For each slide, prepare a labeled microcentrifuge tube with 350 microliters of the lysis buffer containing-beta mercaptoethanol. Use the sections remaining after the LCM to extract RNA.Carefully separate the film from the membrane and lyse the sample by slowly pippetting the lysis buffer onto the section several times. Then, put the lysate samples on dry ice and store them at minus 80 degrees Celsius. When ready to continue, thaw the lysates at room temperature.After this, transfer the lysates from LCM-harvested cells in the collection tubes to new 1.5-milliliter microcentrifuge tubes and extract RNA according to the manufacturer's instructions. In this study, an LCM protocol is developed to obtain sufficient quantity of high quality RNA for gene expression analysis in bone cells of mouse femurs. LCM is performed using an LCM system that uses gravity for sample collection.The yield and integrity of isolated RNA was measured using microcapillary electrophoresis. The difference in RNA quality and quantity obtained using different lysis protocols can be seen here in the representative gel and electropherograms. When the sample is lysed by pippetting up and down in the cap for one minute, it is possible to isolate approximately 8.5 nanograms of RNA from one square millimeter of microdissected bone tissue.The RIN value is 8.60. Alternatively, LCM is performed using an LCM system that uses a defocused laser pulse, which catapults the material into the overhanging adhesive cap. For fresh frozen bones, it is possible to isolate approximately 1.6 nanograms of RNA from one square millimeter of microdissected bone tissue.The RIN value is one. In conclusion, the most important things to remember in this procedure are apply the standing protocol in the correct manner, prevent complete dryout of sections in the sandwich complex, use an appropriate LCM system, and do not exceed the time limits for harvesting the cells. Once you have isolated RNA from the captured cells, you can use the RNA for expression profiling, for example, by RNA seek.Finally, we would like to remind you that xylene and beta-mercaptoethanol are hazardous substances that need to be handled under a hood. In addition, please take appropriate precautions when handling liquid nitrogen and also the cryotome blades.

Research

JoVE Journal

Biology

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