一个单一的鼠标骨骼肌的相邻区Cryosectioning的基因表达和组织学分析
Summary
连续的低温部分被收集，以便利用邻近地区从一个单一的小鼠骨骼肌基因表达测量组织的应用程序和RNA的富集。 30毫克汇集冷冻切片和测量的应用程序之间直接比较 – 高品质的RNA是从20获得。
Abstract
用这种方法，连续冷冻切片被收集，以使对组织的组织学和RNA的富集基因的表达使用相邻区域从单个小鼠骨骼肌既显微镜应用。通常情况下，它是具有挑战性的，实现小骨骼肌样品充分均质化，因为缓冲器的卷可能太低高效研磨应用中，但没有足够的机械破碎，缓冲试剂的肌肉限制渗透的致密组织架构，最终导致低的RNA产量。按照此处所报告的协议，30微米的部分被收集并汇集允许cryosectioning和随后的针均化以机械地扰乱肌肉，增加暴露缓冲渗透的表面积。该技术的主要限制是，它需要一个低温恒温器，它是相对较低的吞吐量。然而，高质量的RNA可从汇集M个小样品中获得uscle冷冻切片，使得这种方法对于许多不同的骨骼肌肉和其他组织访问。此外，这种技术使得能够匹配分析（ 例如，组织病理学和基因表达），以便测量可以跨应用程序，以减少实验的不确定性，并减少所需的复制动物实验，以源为一个小的组织直接比较由单一骨骼肌的相邻区域多个应用程序。
Introduction
该技术的目的是通过不同的方式，如组织学和基因表达，从一个单一的小骨骼肌源组织访问进行多次实验的分析。显微镜应用是最敏感的样品的保存方法，其必须小心地控制冷冻保存期间限制冰晶工件的形成。因此，方法的发展是基于胫骨前肌（TA）肌肉冻部分覆盖在-140℃的液氮冷却的2-甲基丁烷浴为免疫荧光显微镜和基因表达分析源材料嵌入树脂。
用于多样的技术方法相同的源材料，需要的是用于肌内注射的基于实验，其中左和右的肌肉代表不同条件，一是实验和一个控制特别重要。例如，在肌肉再生研究，一亩SCLE与毒素注射引起广泛组织损害而对侧肌肉用作媒介物注射的对照1。同样，对于肌肉疾病的基因治疗研究通常用肌肉注射的基因治疗载体的有效性开始与空载体，无关向量或车辆控制在对侧2进行比较。因此，这是不可能的每个的TA肌肉源到不同的应用程序。
公共策略来处理这个问题是：i）使用不同的肌肉群的每个应用程序，ⅱ）使用额外的小鼠，或iii）切断一块肌肉为每个应用程序的。然而，肌肉群之间相当大的差异，很难将数据从单独的应用程序进行比较，并附加动物增加费用，并且如果其他方法存在不良有道理的。除肌肉解剖后到源不同的应用程序是最好的选择我N多的情况。然而，肌片往往太小使用粉碎液氮或机械研磨技术下均质化2-5。肌肉是填充有细胞外基质和收缩蛋白高度的结构组织，机械均化不足导致的随后的DNA，RNA或蛋白质的低的产率。这里详述该方法允许少量的组织从一个源的肌肉在多个应用程序使用，并cryosectioning和针研磨列入改善了更好的RNA产量机械同质化。
Protocol
所有的动物程序进行格鲁吉亚机构动物护理和使用委员会的大学动物下使用协议A2013 07-016（Beedle）的批准。 1.未固定骨骼肌冷冻保存制备切软木成小方块（约1cm×1厘米）用刀片，用细尖标记物到2-甲基丁烷耐识别源鼠标和肌肉，并进行了很浅的切口在软木写（约1毫米）在整个上表面上。将塑料盖玻片到剪切用于定向组织。重复，直到软木塞准备好要冷冻保存每一个组织。 <l…
Representative Results
肌肉冷冻切片RNA质量高，并提供足够的产量为大多数应用十六骨骼肌RNA制剂分析显示在使用池胫骨前（TA）肌的19.4〜41毫克，从8对照组小鼠表1。两个左（L）和在再生实验中制备与收集的25μl的生理盐水或10μM的心脏毒素的纵向肌内注射后第3天以引起使用方法肌肉损伤肌肉右（R）的TA肌肉先前报道1。如?…
Discussion
为了实现用这种方法最好的结果，保持组织冷冻期间包埋树脂限制在低三分之一或一半的肌肉因为过量的树脂会减缓汇集冷冻切片的集合，并且可以增加在RNA分离包埋树脂污染。另外，针均化期间仔细注意重要的是最大限度地提高产量和最小化堵塞针的概率。该协议可以通过使用一个Luer-Lok接头的注射器，以防止样品的损失，如果在针被阻塞并且需要高压以移去堵塞进行修改。还可以加入具有25或…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Cork | VWR Scientific | 23420-708 | Cut into small squares with a sharp blade. |
| Plastic coverslip | Fisher Scientific | 12-547 | Used to orient the muscle during freezing. |
| Low temperature thermometer | VWR Scientific | 89370-158 | |
| 2-methylbutane | Sigma | M32631-4L | Caution: hazardous chemical. Store in flammable cabinet. |
| Embedding resin: "cryomatrix" | Thermo Fisher Scientific | 6769006 | Other embedding resins can be substituted for cryomatrix. |
| Cryostat | Thermo Fisher Scientific | microm HM550 with disposable blade carrier | Any working cryostat should be sufficient for the protocol. |
| Disposable cryostat blade | Thermo Fisher Scientific | 3052835 | Use an appropriate blade or knife for the cryostat to be used. |
| RNAse decontamination solution: "RNase Zap" | Thermo Fisher Scientific | AM9780 | |
| Analytical balance | Mettler Toledo | XS64 | |
| Paint brush | Daler Rowney | 214900920 | Use to handle cryosections. Can be found with in stores with simple art supplies. |
| Razor blade | VWR Scientific | 55411-050 | |
| Microscope slide | VWR Scientific | 48311600 | |
| RNA organic extraction reagent: TRIzol | Thermo Fisher Scientific | 15596026 | Caution: TRIzol is a hazardous chemical. Note: Only organic extraction reagents are recommended for RNA extraction from skeletal muscle. |
| 18 gauge needle | VWR Scientific | BD305185 | |
| 22 gauge needle | VWR Scientific | BD305155 | |
| 26 gauge needle | VWR Scientific | BD305115 | Optional. Can be used for a third round of sample trituration in the RNA extraction protocol. |
| 1 mL syringe | VWR Scientific | BD309659 | For very high value samples, a luer-lok syringe is recommended (e.g. VWR BD309628). |
| 1-bromo-3-chloropentane (BCP) | Sigma | B9673 | |
| For 70% ethanol in DEPC water: 200 proof alcohol | Decon Laboratories, Inc. | +M18027161M | Mix 35 ml 200 proof alcohol + 15 mL DEPC water. |
| For 70% ethanol in DEPC water: DEPC-treated water | Thermo Fisher Scientific | AM9922 | Mix 35 ml 200 proof alcohol + 15 mL DEPC water. |
| RNA purification kit: PureLink RNA minikit | Thermo Fisher Scientific | 12183018A | Final steps of RNA preparation. |
| DNase/Rnase-free water | Gibco | 10977 | DEPC-treated water can also be used. |
| Spectrophotometer: Nanodrop 2000 | Thermo Fisher Scientific | NanoDrop 2000 | |
| Dnase I | Thermo Fisher Scientific | AM2222 | Treat purified RNA to remove any DNA contamination before downstream appications. |
| Hydrophobic pen | Thermo Fisher Scientific | 8899 | |
| Dulbecco's PBS | Gibco | 14190 | PBS for immunofluorescence protocol. |
| Donkey serum | Jackson ImmunoResearch Laoratories, Inc | 017-000-121 | Rehydrate normal donkey serum stock according to the manufacturer's instructions, then dilute an aliquot to 5% for immunofluorescence. Normal goat serum can also be used. |
| eMHC antibody | University of Iowa Developmental Studies Hybridoma Bank | F1.652 | |
| Collagen VI antibody | Fitzgerald Industries | #70R-CR009x | |
| Donkey anti-rabbit AlexaFluor488 | Thermo Fisher Scientific | A21206 | |
| Goat anti-mouse IgG1 AlexaFluor546 | Thermo Fisher Scientific | A21123 | |
| DAPI (4',6-diamidino-2-phenylindole) | Thermo Fisher Scientific | D1306 | |
| Aqueous mounting media: Permafluor | Thermo Fisher Scientific | TA-030-FM | Only use mounting media designed for fluorescent applications with anti-fade properties. |
| Glass coverslip | VWR Scientific | 16004-314 | Use for mounting slides at the end of immunofluorescence protocl |
| Image analysis software: ImageProExpress | Media Cybernetics, Inc. | Image-Pro Express, or more advanced products | Freeware ImageJ should also work for manual counting. More advanced software with segmentation abiities may allow partial automation of the process. E.g. ImageProPremier. |
| Merge and map section images: Photoshop | Adobe | Photoshop | |
| Cardiotoxin | Sigma | C9659 | 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. |
| reverse transcription kit: Superscript III First-strand synthesis system | Thermo Fisher Scientific | 18080051 | Any validated, high quality reverse transcription reagents can be used. |
| Standard PCR: GoTaq Flexi polymerase system | Promega | M8298 | 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. |
| SYBR green | Thermo Fisher Scientific | S7585 | 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. |
| ROX: SuperROX, 15 mM | BioResearch Technologies, Inc. Novato CA | SR-1000-10 | 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 |
| Real-time PCR | Applied Biosystems | 7900HT |
References
- Foltz, S. J., et al. Abnormal skeletal muscle regeneration plus mild alterations in mature fiber type specification in Fktn-Deficient Dystroglycanopathy Muscular Dystrophy Mice. PLoS One. 11 (1), 0147049 (2016).
- Bartoli, M., et al. Noninvasive monitoring of therapeutic gene transfer in animal models of muscular dystrophies. Gene Ther. 13, 20-28 (2006).
- Ip, W. T., Huggins, C. E., Pepe, S., Delbridge, L. M. Evaluating RNA preparation options for archived myocardial biopsies. Heart Lung Circ. 20 (5), 329-331 (2011).
- Beedle, A. M., et al. Mouse fukutin deletion impairs dystroglycan processing and recapitulates muscular dystrophy. J. Clin. Invest. 122 (9), 3330-3342 (2012).
- Foltz, S. J., et al. Four-week rapamycin treatment improves muscular dystrophy in a fukutin-deficient mouse model of dystroglycanopathy. Skeletal Muscle. 6, 20 (2016).
- Liu, L., Cheung, T. H., Charville, G. W., Rando, T. A. Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting. Nat. Protoc. 10, 1612-1624 (2015).
- Rump, L. V., Asamoah, B., Gonzalez-Escalona, N. Comparison of commercial RNA extraction kits for preparation of DNA-free total RNA from Salmonella cells. BMC Res. Notes. 3, 211 (2010).
- Desjardins, P., Conklin, D. NanoDrop microvolume quantitation of nucleic acids. J. Vis. Exp. (45), e2565 (2010).
- Valdez, M. R., Richardson, J. A., Klein, W. H., Olson, E. N. Failure of Myf5 to support myogenic differentiation without myogenin, MyoD, and MRF4. Dev Biol. 219 (2), 287-298 (2000).
- Pavlath, G. K., Dominov, J. A., Kegley, K. M., Miller, J. B. Regeneration of transgenic skeletal muscles with altered timing of expression of the basic helix-loop-helix muscle regulatory factor MRF4. Am. J. Pathol. 162 (5), 1685-1691 (2003).
- Chomczynski, P., Mackey, K. Substitution of chloroform by bromo-chloropropane in the single-step method of RNA isolation. Anal. Biochem. 225 (1), 163-164 (1995).
- Lee, J. T. Y., Cheung, K. M. C., Leung, V. Y. L. Extraction of RNA from tough tissues with high proteoglycan content by cryosection, second phase separation and high salt precipitation. J. Biol. Methods. 2 (2), 20 (2015).
- Guo, D., Catchpoole, D. R. Isolation of intact RNA following cryosections of archived frozen tissue. BioTechniques. 34, 48-50 (2003).
- Goodpaster, T., Randolph-Habecker, J. A flexible mouse-on-mouse immunohistochemical staining technique adaptable to biotin-free reagents, immunofluorescence, and multiple antibody staining. J. Histochem. Cytochem. 62 (3), 197-204 (2014).
- Ikezawa, M., Minami, N., Takahashi, M., Goto, Y., Miike, T., Nonaka, I. Dystrophin gene analysis of 130 patients with Duchenne muscular dystrophy with a special reference to muscle mRNA analysis. Brain Dev. 20 (3), 165-168 (1998).
- Majumdar, G., Vera, S., Elam, M. B., Raghow, R. A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle. Anal. Biochem. 474, 25-27 (2015).
- Espina, V., Heiby, M., Pieroban, M., Liotta, L. A. Laser capture microdissection technology. Expert Rev Mol Diagnostics. 7 (5), 647-657 (2007).
Cite This Article
Beedle, A. M. Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses. J. Vis. Exp. (118), e55058, doi:10.3791/55058 (2016).