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.

모든 동물의 절차는 동물 사용 프로토콜 A2013 07-016 (비들)에서 조지아 기관 동물 관리 및 사용위원회의 대학에 의해 승인되었다. 미 정착 골격근 1. 냉동 보존 <ol><li> 예비 <ol><li> 약 (작은 사각형 면도날과 (약 1cm X 1cm)에 코르크를 잘라 소스 마우스와 근육을 식별하고, 매우 얕은 컷을 만들기 위해 2 메틸 부탄에 내성이 좋은 팁 마커 코르크에 쓰기 상면 전역 1mm). 조직을 배향에 사용할 컷에 ?...

<ol>
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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>

과잉 수지가 풀 저온부의 수집 속도가 느려집니다 및 RNA 분리에 수지 오염을 포함 증가시킬 수 있기 때문에이 방법으로 최상의 결과를 얻으려면, 조직 냉동 보존하는 동안 근육의 낮은 세 번째 또는 절반으로 제한 수지를 포함 유지. 또한, 바늘 균질화 동안 세심한주의 수율을 최대화하고, 바늘 막힘 가능성을 최소화하는 것이 중요하다. 프로토콜은 니들이 차단 방해물을 제거하기 위해 높은 압력...

이 기술의 목적은 하나의 작은 골격근 조직 소스로부터 액세스 같은 조직 학적 및 유전자 발현과 같은 다른 양상, 여러 실험을 분석하는 것이다. 현미경 애플리케이션 신중 동결 동안 얼음 결정 생성물의 형성을 제한하도록 제어 되어야만 보존 방법을 시료에 가장 민감하다. 따라서, 분석법 개발을 경골근에 기초한다 (TA) 근 부분적으로 면역 형광 현미경 유전자 발현 양 분석을위한 원료로서 -140 ° C 액체 질소 냉각 -2- 메틸 부탄 욕에서 수지 매립 덮여 냉동. 다양한 기술적 접근법 동일한 원료를 사용할 필요가 좌우 근육 상이한 조건 실험 하나는 제어를 나타내는 근육 주사 기반 실험을 위해 특히 중요하다. 예를 들어, 근육의 재생 과정에서, 하나의 MUscle는 반대측 근육 차량 주입 제어 한 역할을하면서 광범위한 조직 손상을 유발하는 독소를 주사한다. 마찬가지로, 근육 질환에 대한 유전자 치료 연구는 일반적으로 반대쪽이 빈 벡터, 벡터 또는 비 관련 차량 제어와 비교되는 근육 내 주사에 의한 유전자 치료 벡터의 유효성 시작한다. 따라서, 다른 어플리케이션에 각각 TA 근육을 소싱 할 수 없다. 이러한 문제점을 해결하기위한 일반적인 전략은 : ⅰ) ⅱ) 추가 마우스를 사용하거나, ⅲ) 각각의 애플리케이션에 대한 근육의 일부를 잘라, 어플리케이션마다 다른 근육 그룹을 사용. 그러나, 근육 그룹간에 상당한 차이가 어려운 개별 애플리케이션으로부터 데이터를 비교할 수 있도록, 추가 동물 비용을 증가시키고, 다른 대안이있을 경우 제대로 정렬된다. 다른 응용 프로그램을 소스로 절개 한 후 근육을 나누는 것은 최선의 선택의 난입니다n은 많은 경우. 단, 근육의 조각들은 균질화 2-5 액체 질소 또는 기계적 연마 기술에 따라 분쇄를 사용하기에는 너무 작다. 근육 세포 외 매트릭스 단백질과 수축 포장 높은 조직 구조이기 때문에, 불충분 한 기계적 균질화 이후 DNA, RNA 또는 단백질의 낮은 수율로 이끈다. 여기에 자세한 방법은 여러 응용 프로그램에서 사용하기위한 하나의 소스 근육에서 조직의 소량을 허용하고, 냉동 절편 바늘 분쇄의 포함은 더 나은 RNA 수율 기계적 균질화을 향상시킨다.

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		<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

이 방법으로, 연속 저온부는 하나의 마우스 골격 근육에서 인접 영역을 사용하여 유전자 발현을위한 조직 조직학 및 RNA의 농축 모두 현미경 응용 프로그램을 사용하기 위해 수집됩니다. 전형적으로, 그것은, 버퍼 볼륨이 효율적으로 연마 애플리케이션 너무 낮을 수 있기 때문에 아직 충분한 기계적 중단없이 작은 골격근 샘플의 적절한 균질화를 달성하는 도전 버퍼 시약 근육 제한 침투 치밀한 조직 구조는 궁극적으로 저 RNA 수율을 야기. 여기에보고 된 프로토콜에 따라 30 ㎛의 섹션을 수집하고 버퍼 침투 노출 표면적을 증가 저온 절편 이후 니들 균질화가 근육을 방해 기계적하게 풀링. 이 기술의 주요 제한은 저온 유지가 필요하다고하고, 이는 상대적으로 낮은 스루풋이다. 그러나, 고품질의 RNA를 풀링 m 작은 샘플로부터 얻을 수있다uscle 저온부는 여러 가지 골격 근육과 다른 조직이 방법은 액세스 할 수 있도록. 또한,이 기술은 유사한 가능 분석 (예, 조직 병리학 및 유전자 발현) 측정치가 직접 실험 불확실성을 감소시키고위한 작은 조직 소스 필요 복제 성 동물 실험을 감소시키는 애플리케이션에서 비교 될 수 있도록 하나의 골격근의 인접 영역에서 여러 응용 프로그램.

근육을 동결 절단 (Cryosections)의 RNA는 품질이 높고, 대부분의 응용 프로그램에 충분한 수율을 제공합니다 여섯 골격근 RNA 제제의 분석은 8 대조군 마우스로부터 풀링 경골근 (TA) 근 19.4 41 mg의 사용 표 1에 나타낸다. 모두 좌측 (L) 및 우측 (R) TA 근육이 방법을 이용하여 근육 손상을 일으킬 삼일 식염수 또는 10 μM의 cardi...

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

연속 크라이 섹션은 단일 마우스 골격근에서 인접 영역을 이용한 유전자 발현 측정 학적 어플리케이션 및 RNA의 농축을 가능하게 수집된다. 직접 애플리케이션에서 비교 풀링 저온부 및 측정 30mg을 - 고품질 RNA를 (20)로부터 얻어진다.

유전자 발현 및 조직 학적 분석을위한 단일 마우스 골격근의 연속 지역의 냉동 절편

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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Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

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

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

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

유전자 발현 연구를 단순화하는 자동 셀 카운터를 사용하여 : Namalwa 셀에서 IL - 4 종속 유전자 발현의 siRNA의 최저

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 ...

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

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In vitro Reconstitution of the Active T. castaneum Telomerase

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

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

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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

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