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.

<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>

The author declares that she has no competing financial interests.

כדי להשיג את התוצאות הטובות ביותר עם שיטה זו, לשמור הטבעת השרף המוגבל לנמוך השליש או המחצית של השריר במהלך cryopreservation רקמות בגלל שרף עודף יאט את האוסף של cryosections ונקווה ועלול להגדיל הטבעת זיהום שרף בידוד RNA. כמו כן, תשומת לב קפדנית במהלך המגון מחט חשוב למקסם את התשואה ולה?...

מטרת טכניקה זו היא להפוך ניתוחים ניסיוניים מרובים על ידי שיטות שונות, כגון ביטוי היסטולוגיה גן, נגישות מכל רקמה ממקור שרירי שלד אחת קטנה. יישומים מיקרוסקופים הם הרגישים ביותר כדי לטעום דרכי שימור, אשר חייב להיות מבוקר בקפידה כדי להגביל את ההיווצרות של חפצי גביש הקרח במהלך cryopreservation. לפיכך, פיתוח שיטה מבוסס על קדמי tibialis (ת&quot;א) שריר קפוא מכוסה חלקית עם ההטבעה שרפה באמבט חנקן מקורר נוזל -140 ° C 2-methylbutane כחומר המקור הוא מיקרוסקופיה immunofluorescence ביטוי גני המנתח. הצורך להשתמש באותו מקור חומר עבור גישות טכניות מגוונות חשוב במיוחד עבור ניסויים מבוססי זריקה תוך שרירית שבו השרירים ימין ועל שמאל מייצגים תנאים שונים, אחד ניסוי ובקרה אחד. לדוגמה, במחקרים התחדשות שריר, mu אחדscle מוזרק עם רעלן לגרום נזק לרקמות נרחבת תוך השריר הנגדי משמש כביקורת הרכב מוזרק 1. בדומה לכך, מחקרי ריפוי גנטי להפרעות שריר בדרך כלל מתחילים עם אימות של וקטור הריפוי הגנטי על ידי זריקה תוך שרירית להיות לעומת וקטור ריק, וקטור קשור או שליטה ברכב בצד הנגדי 2. לכן, אין זה אפשרי למקור כל שריר ת&quot;א ליישום אחר. אסטרטגיות נפוצות להתמודד עם בעיה זו הן: i) להשתמש קבוצת שרירים שונה עבור כל יישום, ii) להשתמש עכברים נוספים, או iii) כדי לחתוך חתיכה של השריר עבור כל יישום. עם זאת, הבדלים משמעותיים בין קבוצות השרירות להקשות להשוות נתונים מיישומים נפרדים, וחיות נוספות להגדיל הוצאה והם גרועים מוצדקים אם קיימות חלופות אחרות. חלוקת השרירים לאחר דיסקציה למקור יישומים שונים היא i האפשרות הטוב ביותרבמקרים רבים n. עם זאת, את החלקים שרירים הם בדרך כלל קטנים מדי לשימוש אִבּוּק תחת חנקן נוזלי או טכניקות שחיקה מכנית עבור המגון 2-5. כמו שריר היא רקמה מבנית מאוד עמוסה חלבוני מטריקס התכווצות תאיים, המגון מכאני לקוי מוביל בתשואה נמוכה של DNA, RNA או חלבון שלאחר מכן. השיטה המפורטת כאן מאפשרת כמויות קטנות של רקמת שריר מקור אחד לשימוש ביישומים מרובים, וההכללה של טחינה דקה cryosectioning ומחט משפר המגון מכנה תשואת RNA טובה.

<table border="0" cellpadding="0" cellspacing="0" width="637">
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		<col />
		<col />
		<col />
	</colgroup>
	<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

באמצעות שיטה זו, ברציפות cryosections נאספים כדי לאפשר הן יישומים במיקרוסקופ לצורך היסטולוגיה רקמה והעשרה של רנ&quot;א עבור ביטוי גנים באמצעות אזורים סמוכים מתוך שרירי השלד יחיד העכבר. בדרך כלל, אתה עשוי להתקשות להשיג הומוגניזציה נאותה דגימות שריר שלד קטנות בגלל כרכי חיץ עשויים להיות נמוכים מדי עבור יישומי שחיקה יעילים, עדיין ללא הפרעה מכאנית מספיק, ארכיטקטורת הרקמה הצפופה של חדירה לתחום שריר של ריאגנטים חיץ, גורמת בסופו של דבר תשואת RNA נמוכה. על ידי ביצוע הפרוטוקול שדווח כאן, 30 מיקרומטר חלקים נאספים ונקווים המאפשרים cryosectioning ויצירת הומוגניות מחט לאחר מכנית לשבש את השריר, הגדלת שטח הפנים החשוף לחדירת חיץ. המגבלות העיקריות של הטכניקה הן כי זה דורש cryostat, וזה יחסית תפוקה נמוכה. עם זאת, RNA באיכות גבוהה ניתן להשיג דגימות קטנות של מטר ויקווהcryosections uscle, מה שהופך שיטה זו נגישה עבור שרירי שלד ורקמות אחרות רבים ושונים. יתר על כן, טכניקה זו מאפשרת ניתוחים מתאימים (למשל, histopathology רקמות ביטוי גנים) מאזורים סמוכים של שרירי שלד יחידים כך שמדידות ניתן להשוות באופן ישיר בין יישומים כדי לצמצם את אי הוודאות ניסיונית להפחית ניסויים בבעלי חיים בשכפול הדנ&quot;א צורך למקור רקמה קטנה עבור יישומים מרובים.

RNA השרירים cryosection הוא באיכות גבוהה ומספק תשואה מספיק עבור מרבית היישומים ניתוח של שש עשר הכנות RNA שרירי שלד מוצג בלוח 1 באמצעות 19.4 כדי 41 מ&quot;ג קדמי tibialis נקווה (ת&quot;א) שריר מ 8 עכברים שליטים....

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

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

קריו-סעיפים רצופים נאספים כדי לאפשר יישומים היסטולוגית והעשרה של RNA למדידות ביטוי גנים באמצעות אזורים סמוכים מתוך שרירי שלד יחיד עכבר. RNA באיכות גבוהה מתקבל מ 20 - 30 מ&quot;ג של cryosections ומדידות ונקווה מושווים ישירות בין יישומים.

Cryosectioning של רציף אזורים של שרירי שלד עכבר אחת על ביטוי גנים ועל ניתוח היסטולוגית

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

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

15.6K Views.  University of Georgia.  קריו-סעיפים רצופים נאספים כדי לאפשר יישומים היסטולוגית והעשרה של RNA למדידות ביטוי גנים באמצעות אזורים סמוכים מתוך שרירי שלד יחיד עכבר. RNA באיכות גבוהה מתקבל מ 20 - 30 מ"ג של cryosections ומדידות ונקווה מושווים ישירות בין יישומים. 

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

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

הכנת ראשי בתרביות תאים hematopoietic ממח העצם Murine עבור electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

שימוש ג&#39;ין Pulser מערכת MXcell electroporation כדי Transfect תאים ראשוניים עם יעילות גבוהה

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

שימוש מונה תא אוטומטי כדי לפשט את הביטוי מחקרים ג&#39;ין: siRNA מציאה הביטוי IL 4-Gene תלויים תאים Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

למבוגרים עובריים שרירי השלד Microexplant התרבות בידוד בתאי גזע שרירי השלד

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

מדורים פרפין-Embedded קפואים של תסיסנית סקס שרירים

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

ב הכינון במבחנה של ט פעילים castaneum שהטלומרז

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

בידוד ותרבות של Myofibers פרט ותאי הלווין שלהם משרירי שלד למבוגרים

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

ניתוח של גלובל RNA סינתזה ברמת התא בודד הבאות היפוקסיה

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

Triage רקמות ומקפיאים למודלים של מחלות שרירי שלד

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Cryosectioning של רציף אזורים של שרירי שלד עכבר אחת על ביטוי גנים ועל ניתוח היסטולוגית

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