Name: cryosectioning of contiguous regions of a single mouse skeletal muscle for gene expression and histological analyses
Uploaded: 2016-12-12T15:00:00
Description: Watch this Scientific Journal Video about Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses at JoVE.com

Madison Grant, Steven Foltz, Halie Zastre and Junna Luan provided technical assistance. Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under award number AR065077. The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.

All animal procedures were approved by the University of Georgia Institutional Animal Care and Use Committee under animal use protocol A2013 07-016 (Beedle).
1. Cryopreservation of Unfixed Skeletal Muscle
<ol>
	<li>Preparation
	<ol>
		<li>Cut cork into small squares (approximately 1 cm x 1 cm) with a razor blade, write on the cork with a fine tip marker that is resistant to 2-methylbutane to identify the source mouse and muscle, and make a very shallow cut (approximately 1 mm) across the top surface. Insert a pl...

<ol>
	<li>Foltz, S. J., 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=Abnormal+skeletal+muscle+regeneration+plus+mild+alterations+in+mature+fiber+type+specification+in+Fktn-Deficient+Dystroglycanopathy+Muscular+Dystrophy+Mice.">Abnormal skeletal muscle regeneration plus mild alterations in mature fiber type specification in Fktn-Deficient Dystroglycanopathy Muscular Dystrophy Mice.</a> PLoS One. 11 (1), 0147049 (2016).</li><li>Bartoli, 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=Noninvasive+monitoring+of+therapeutic+gene+transfer+in+animal+models+of+muscular+dystrophies.">Noninvasive monitoring of therapeutic gene transfer in animal models of muscular dystrophies.</a> Gene Ther. 13, 20-28 (2006).</li><li>Ip, W. T., Huggins, C. E., Pepe, S., Delbridge, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+RNA+preparation+options+for+archived+myocardial+biopsies.">Evaluating RNA preparation options for archived myocardial biopsies.</a> Heart Lung Circ. 20 (5), 329-331 (2011).</li><li>Beedle, A. 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=Mouse+fukutin+deletion+impairs+dystroglycan+processing+and+recapitulates+muscular+dystrophy.">Mouse fukutin deletion impairs dystroglycan processing and recapitulates muscular dystrophy.</a> J. Clin. Invest. 122 (9), 3330-3342 (2012).</li><li>Foltz, S. J., 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=Four-week+rapamycin+treatment+improves+muscular+dystrophy+in+a+fukutin-deficient+mouse+model+of+dystroglycanopathy.">Four-week rapamycin treatment improves muscular dystrophy in a fukutin-deficient mouse model of dystroglycanopathy.</a> Skeletal Muscle. 6, 20 (2016).</li><li>Liu, L., Cheung, T. H., Charville, G. W., Rando, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+skeletal+muscle+stem+cells+by+fluorescence-activated+cell+sorting.">Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting.</a> Nat. Protoc. 10, 1612-1624 (2015).</li><li>Rump, L. V., Asamoah, B., Gonzalez-Escalona, N. <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+commercial+RNA+extraction+kits+for+preparation+of+DNA-free+total+RNA+from+Salmonella+cells.">Comparison of commercial RNA extraction kits for preparation of DNA-free total RNA from Salmonella cells.</a> BMC Res. Notes. 3, 211 (2010).</li><li>Desjardins, P., Conklin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoDrop+microvolume+quantitation+of+nucleic+acids.">NanoDrop microvolume quantitation of nucleic acids.</a> J. Vis. Exp. (45), e2565 (2010).</li><li>Valdez, M. R., Richardson, J. A., Klein, W. H., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+of+Myf5+to+support+myogenic+differentiation+without+myogenin,+MyoD,+and+MRF4.">Failure of Myf5 to support myogenic differentiation without myogenin, MyoD, and MRF4.</a> Dev Biol. 219 (2), 287-298 (2000).</li><li>Pavlath, G. K., Dominov, J. A., Kegley, K. M., Miller, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+transgenic+skeletal+muscles+with+altered+timing+of+expression+of+the+basic+helix-loop-helix+muscle+regulatory+factor+MRF4.">Regeneration of transgenic skeletal muscles with altered timing of expression of the basic helix-loop-helix muscle regulatory factor MRF4.</a> Am. J. Pathol. 162 (5), 1685-1691 (2003).</li><li>Chomczynski, P., Mackey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substitution+of+chloroform+by+bromo-chloropropane+in+the+single-step+method+of+RNA+isolation.">Substitution of chloroform by bromo-chloropropane in the single-step method of RNA isolation.</a> Anal. Biochem. 225 (1), 163-164 (1995).</li><li>Lee, J. T. Y., Cheung, K. M. C., Leung, V. Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+RNA+from+tough+tissues+with+high+proteoglycan+content+by+cryosection,+second+phase+separation+and+high+salt+precipitation.">Extraction of RNA from tough tissues with high proteoglycan content by cryosection, second phase separation and high salt precipitation.</a> J. Biol. Methods. 2 (2), 20 (2015).</li><li>Guo, D., Catchpoole, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+intact+RNA+following+cryosections+of+archived+frozen+tissue.">Isolation of intact RNA following cryosections of archived frozen tissue.</a> BioTechniques. 34, 48-50 (2003).</li><li>Goodpaster, T., Randolph-Habecker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flexible+mouse-on-mouse+immunohistochemical+staining+technique+adaptable+to+biotin-free+reagents,+immunofluorescence,+and+multiple+antibody+staining.">A flexible mouse-on-mouse immunohistochemical staining technique adaptable to biotin-free reagents, immunofluorescence, and multiple antibody staining.</a> J. Histochem. Cytochem. 62 (3), 197-204 (2014).</li><li>Ikezawa, M., Minami, N., Takahashi, M., Goto, Y., Miike, T., Nonaka, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophin+gene+analysis+of+130+patients+with+Duchenne+muscular+dystrophy+with+a+special+reference+to+muscle+mRNA+analysis.">Dystrophin gene analysis of 130 patients with Duchenne muscular dystrophy with a special reference to muscle mRNA analysis.</a> Brain Dev. 20 (3), 165-168 (1998).</li><li>Majumdar, G., Vera, S., Elam, M. B., Raghow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+streamlined+protocol+for+extracting+RNA+and+genomic+DNA+from+archived+human+blood+and+muscle.">A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle.</a> Anal. Biochem. 474, 25-27 (2015).</li><li>Espina, V., Heiby, M., Pieroban, M., Liotta, L. A. <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+technology.">Laser capture microdissection technology.</a> Expert Rev Mol Diagnostics. 7 (5), 647-657 (2007).</li></ol>

To achieve best results with this method, keep embedding resin restricted to the lower third or half of the muscle during tissue cryopreservation because excess resin will slow the collection of the pooled cryosections and may increase embedding resin contamination in the RNA isolation. Also, careful attention during needle homogenization is important to maximize yield and minimize the probability of clogging the needle. The protocol may be modified by using a Luer-Lok syringe to protect against sample loss if the needle...

The goal of this technique is to make multiple experimental analyses by different modalities, such as histology and gene expression, accessible from a single small skeletal muscle source tissue. Microscopy applications are the most sensitive to sample preservation methods, which must be carefully controlled to limit the formation of ice crystal artifacts during cryopreservation. Thus, method development is based on the tibialis anterior (TA) muscle frozen partially covered with embedding resin in a -140 &#176;C liquid nitrogen-cooled 2-methylbutane bath as the source material for both immunofluorescence microscopy and gene expression analyses.
The need to use the same source material for diverse technical approaches is particularly important for intramuscular injection-based experiments where the left and right muscles represent different conditions, one experimental and one control. For example, in muscle regeneration studies, one muscle is injected with a toxin to cause widespread tissue damage while the contralateral muscle serves as a vehicle-injected control1. Similarly, gene therapy studies for muscle disorders typically begin with validation of the gene therapy vector by intramuscular injection to be compared with empty vector, unrelated vector or vehicle control on the contralateral side2. Therefore, it is not possible to source each TA muscle to a different application.
Common strategies to deal with this issue are: i) to use a different muscle group for each application, ii) to use additional mice, or iii) to cut off a piece of the muscle for each application. However, substantial differences between muscle groups make it difficult to compare data from separate applications, and additional animals increase expense and are poorly justified if other alternatives exist. Dividing the muscle after dissection to source different applications is the best option in many cases. However, the muscle pieces are often too small to use pulverization under liquid nitrogen or mechanical grinding techniques for homogenization2-5. As muscle is a highly structural tissue packed with extracellular matrix and contractile proteins, inadequate mechanical homogenization leads to a low yield of subsequent DNA, RNA or protein. The method detailed here allows small quantities of tissue from one source muscle for use in multiple applications, and the inclusion of cryosectioning and needle trituration improves mechanical homogenization for better RNA yield.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Cork</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">23420-708</td>
			<td style="width:259px;">Cut into small squares with a sharp blade.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Plastic coverslip</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:115px;">12-547</td>
			<td style="width:259px;">Used to orient the muscle during freezing.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Low temperature thermometer</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">89370-158</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">2-methylbutane</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:115px;">M32631-4L</td>
			<td style="width:259px;">Caution: hazardous chemical. Store in flammable cabinet.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Embedding resin: &#34;cryomatrix&#34;</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">6769006</td>
			<td style="width:259px;">Other embedding resins can be substituted for cryomatrix.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">Cryostat</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">microm HM550 with disposable blade carrier</td>
			<td style="width:259px;">Any working cryostat should be sufficient for the protocol.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Disposable cryostat blade</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">3052835</td>
			<td style="width:259px;">Use an appropriate blade or knife for the cryostat to be used.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:135px;">RNAse decontamination solution: &#34;RNase Zap&#34;</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">AM9780</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">Analytical balance</td>
			<td style="width:128px;">Mettler Toledo</td>
			<td style="width:115px;">XS64</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">Paint brush</td>
			<td style="width:128px;">Daler Rowney</td>
			<td style="width:115px;">214900920</td>
			<td style="width:259px;">Use to handle cryosections. Can be found with in stores with simple art supplies.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">Razor blade</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">55411-050</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">Microscope slide</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">48311600</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:135px;">RNA organic extraction reagent: TRIzol</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">15596026</td>
			<td style="width:259px;">Caution: TRIzol is a hazardous chemical. Note: Only organic extraction reagents are recommended for RNA extraction from skeletal muscle.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">18 gauge needle</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">BD305185</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">22 gauge needle</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">BD305155</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">26 gauge needle</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">BD305115</td>
			<td style="width:259px;">Optional. Can be used for a third round of sample trituration in the RNA extraction protocol.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">1 mL syringe</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">BD309659</td>
			<td style="width:259px;">For very high value samples, a luer-lok syringe is recommended (e.g. VWR BD309628).</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">1-bromo-3-chloropentane (BCP)</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:115px;">B9673</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">For 70% ethanol in DEPC water: 200 proof alcohol</td>
			<td style="width:128px;">Decon Laboratories, Inc.</td>
			<td style="width:115px;">+M18027161M</td>
			<td style="width:259px;">Mix 35 ml 200 proof alcohol + 15 mL DEPC water.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">For 70% ethanol in DEPC water: DEPC-treated water</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">AM9922</td>
			<td style="width:259px;">Mix 35 ml 200 proof alcohol + 15 mL DEPC water.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">RNA purification kit: PureLink RNA minikit</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">12183018A</td>
			<td style="width:259px;">Final steps of RNA preparation.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">DNase/Rnase-free water&#160;</td>
			<td style="width:128px;">Gibco</td>
			<td style="width:115px;">10977</td>
			<td style="width:259px;">DEPC-treated water can also be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Spectrophotometer: Nanodrop 2000</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">NanoDrop 2000</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">Dnase I</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">AM2222</td>
			<td style="width:259px;">Treat purified RNA to remove any DNA contamination before downstream appications.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Hydrophobic pen</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">8899</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">Dulbecco&#39;s PBS</td>
			<td style="width:128px;">Gibco</td>
			<td style="width:115px;">14190</td>
			<td style="width:259px;">PBS for immunofluorescence protocol.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:135px;">Donkey serum</td>
			<td style="width:128px;">Jackson ImmunoResearch Laoratories, Inc</td>
			<td style="width:115px;">017-000-121</td>
			<td style="width:259px;">Rehydrate normal donkey serum stock according to the manufacturer&#39;s instructions, then dilute an aliquot to 5% for immunofluorescence.&#160; Normal goat serum can also be used.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:135px;">eMHC antibody</td>
			<td style="width:128px;">University of Iowa Developmental Studies Hybridoma Bank</td>
			<td style="width:115px;">F1.652</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Collagen VI antibody</td>
			<td style="width:128px;">Fitzgerald Industries</td>
			<td style="width:115px;">#70R-CR009x</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Donkey anti-rabbit AlexaFluor488</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">A21206</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Goat anti-mouse IgG1 AlexaFluor546</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td>A21123</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">DAPI (4&#39;,6-diamidino-2-phenylindole)</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">D1306</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">Aqueous mounting media: Permafluor</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">TA-030-FM</td>
			<td style="width:259px;">Only use mounting media designed for fluorescent applications with anti-fade properties.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Glass coverslip</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">16004-314</td>
			<td style="width:259px;">Use for mounting slides at the end of immunofluorescence protocl</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:135px;">Image analysis software: ImageProExpress</td>
			<td style="width:128px;">Media Cybernetics, Inc.</td>
			<td style="width:115px;">Image-Pro Express, or more advanced products</td>
			<td style="width:259px;">Freeware ImageJ should also work for manual counting. More advanced software with segmentation abiities may allow partial automation of the process.&#160; E.g. ImageProPremier.&#160;&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">Merge and map section images: Photoshop</td>
			<td style="width:128px;">Adobe</td>
			<td style="width:115px;">Photoshop</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:135px;">Cardiotoxin</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:115px;">C9659</td>
			<td style="width:259px;">Sigma C9659 has been discontinued. Other options for cardiotoxin are EMD Millipore #217503; American Custom Chemicals Corp. # BIO0000618; or Ge Script # RP17303; but these have not been validated.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:135px;">reverse transcription kit: Superscript III First-strand synthesis system</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">18080051</td>
			<td style="width:259px;">Any validated, high quality reverse transcription reagents can be used.</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;">Standard PCR: GoTaq Flexi polymerase system</td>
			<td style="width:128px;">Promega</td>
			<td style="width:115px;">M8298</td>
			<td style="width:259px;">Any validated, high quality Taq polymerase system can be used. If DNA sequencing is to be used for any application downstream of the PCR, then a high fidelity PCR system should be used instead.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:135px;">SYBR green</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">S7585</td>
			<td style="width:259px;">For use in qPCR when not using a dedicated qPCR master mix. Use with SuperROX (for Applied Biosystems instruments) and GoTaq Flexi polymerase and buffers.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:135px;">ROX: SuperROX, 15 mM</td>
			<td style="width:128px;">BioResearch Technologies, Inc. Novato CA</td>
			<td style="width:115px;">SR-1000-10</td>
			<td style="width:259px;">SuperROX is more stable in the PCR reaction, so it is preferred for use as a qPCR passive reference dye over ROX (carboxy-X-rhodamine). For qPCR with Applied Biosystems instruments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Real-time PCR</td>
			<td style="width:128px;">Applied Biosystems</td>
			<td style="width:115px;">7900HT</td>
			<td style="width:259px;"></td>
		</tr>
	</tbody>
</table>

cryosectioning of contiguous regions of a single mouse skeletal muscle for gene expression and histological analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Muscle cryosection RNA is high in quality and provides sufficient yield for most applications
Analyses of sixteen skeletal muscle RNA preparations are shown in Table 1 using 19.4 to 41 mg of pooled tibialis anterior (TA) muscle from 8 control mice. Both left (L) and right (R) TA muscles were prepared in regeneration experiments with muscles collected 3 days after longitudinal intramuscular injection of 25 &#956;l o...

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Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

The overall goal of this procedure is to enable analysis of histological and molecular parameters from adjacent sections of a single tissue. With this method, we can help to answer key questions in the field skeletal muscle biology and disease. Such as, what are the regenerative effects after muscular injury, and what are the effects of viral gene delivery by intramuscular injection?The advantage of this method is that histological and molecular data can be directly correlated from a single tissue. That makes more effective data analysis and better use of experimental tissues. Five minutes prior to completing the dissections, cool the cryopreservation bath.When the tissue samples are ready to be embedded, first apply embedding resin to the lower third to half of the tissue, where the muscle meets the cork. This should be done neatly to reduce resin interference with the downstream steps. Then, immediately drop the cork into the 2-Methylbutane bath cooled to negative 140 degrees Celsius.Include up to eight tissue preparations per batch. In the bath, stir the tissues for 30 seconds, scraping the bottom of the beaker to prevent the tissue corks from freezing into the solution. After 30 seconds, using a metal tool, pull a tissue cork from the 2-Methylbutane.Then, quickly remove the supportive cover slip. Dab the excess 2-Methylbutane back into the bath, and then drop the tissue cork into the outer nitrogen bath. After all the tissues have been transferred into the liquid nitrogen, move them to a container and store at negative 80 degrees Celsius.After preparing the cryostat and mounting a tissue sample, use the coarse and fine controls to advance the specimen up to the blade so it touches the blade. Reset the sum of the section thicknesses to zero at this point. Next, set the cryostat to the trim function with section thickness at 30 microns.And cut and discard sections up to the desired tissue depth. To efficiently collect cryosections for RNA extraction, first open the collection tube and place it near the blade carrier. There, use a pre-cooled, clean brush to pick up each section and transfer it to the tube.Pooled 30-micron mouse tibialis anterior sections taken from 400 to 4, 000 micron tissue depth typically yield 25 to 50 milligrams of muscle. If embedding resin surrounds the cryosection, lock the hand-brake and use a razor blade to shave off small pieces of resin until there is only a thin layer around the top of the muscle. Always cut resin with the blade angled away from the muscle.A thin layer of embedding resin does not substantially impair the downstream RNA extraction. If thicker embedding resin is present, use brushes to tease it away from the muscle before moving the cryosection into the collection tube. During the sectioning process, when histological sections are desired, reset the slice thickness to seven microns using the cryostat controls.Then, cut and discard four to seven sections to obtain a consistent, even tissue surface and make note of this tissue depth. Now, cut a good section and orient it on the surface of the blade carrier. Using a room-temperature microscope slide, pick up the section and return the slide to room temperature.Collect all the desired sections and then make note of the final tissue depth. After completing the sectioning, quickly record the weight of the pooled cryosections for RNA isolation. Immediately return the tube to the cryostat chamber to maintain the temperature of the sections near negative 20 degrees Celsius.Begin by quickly transferring the pooled cryosections to ice, and adding approximately one millileter of organic RNA extraction reagent per 50 milligrams of cryosections. The sooner the reagent is applied, the less the RNA degrades. Next using a one milliliter syringe with an 18-gauge needle, homogenize the cryosections without making too many bubbles by repeatedly drawing up a solution, and ejecting it onto the tube walls.Use the needle tip to disperse clumped pieces. After every five strokes, briefly return the sample to the ice to keep it cool. Once homogenized, use a 22-gauge needle to carefully triturate the sample using five more strokes.Then return the sample to ice. Repeat this process three or four times to achieve a very finely dispersed tissue homogeny. Once the final homogenate is made, let it incubate for five minutes at room temperature to disrupt molecular complexes.Next, in a fume hood, add 0.1 milliliter of BCP per milliliter of RNA isolation reagent. Then, shake the tube vigorously for 15 seconds. Do not use a vortex.Then, incubate the sample at room temperature for two to three minutes. Following the brief incubation, centrifuge the sample for 15 minutes at 12, 000g and four degrees Celsius. Then, carefully collect the clear upper phase containing the RNA.Transfer it to a clean tube, and add an equal volume of 70%ethanol, made up in deep seawater. After briefly vortexing the tube, let it incubate for 10 minutes at room temperature and proceed by following the text protocol. Skeletal muscle RNA preparations were made from the mouse tibialis anterior.Three days before, some muscles were injected with 10 micromolar cardiotoxin and others with saline. The purity and yield of the RNA from these samples was excellent. RNA yield was substantially higher in tibialis anterior muscles after toxin injury.Tissue integrity and RNA production could factor into this observation. The persistence of the prepared RNA quality was assessed using one microliter of RNA that had been stored at negative 20 degrees Celsius for 18 months. Prominent 18S and 28S ribosomal RNA bands are still evident in samples demonstrating high RNA quality.Seven-micron sections were stained for expression of embryonic myosin heavy chain, seen in red. To detect regenerating fibers, stained for collagen six, seen in green, which outlines muscle fibers and stained with dappy, in blue, to observe the nuclei. When there is a poor toxin injection, the injury appears minimal.A successful toxin injection results in a much larger region of the targeted muscle compartment being affected. The degree of injury could be used as inclusion criteria for the RNA analysis. Once mastered, cutting sections for RNA extraction and histological slide, can be completed in under half an hour for a single tissue.Following this procedure, other histological of immunofluorescent stains can be followed to answer a wide variety of questions. Pooled cryosections can also be used to collect DNA or protein.

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