从小鼠胰腺RNA提取：核糖核酸酶丰富的组织
Summary
We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.
Abstract
Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.
Introduction
需要常规的分子生物学实验，如Northern印迹9，定量RT-PCR检测1或基因表达谱5与诚信度高的RNA分离。大多数现代的RNA分离方法，是根据对硫氰酸胍协议2，修改3。硫氰酸胍是一种强的蛋白质变性剂，并有效地破坏大多数条件下核糖核酸酶的活性。 Chomczynski和Sacchi 3的流行的方法结合到苯酚的硫氰酸胍裂解溶液，减少了隔离时间约4小时。许多市售的RNA提取试剂基于Chomczynski和Sacchi方法3。
核糖核酸酶丰富的组织如人类或小鼠胰腺呈现为分离RNA一个额外的挑战。小鼠胰腺可含有高达75毫克核糖核酸酶6的其中一些将被释放超级碗造成组织自溶胰腺组织克中断。的异硫氰酸胍协议的修改已经被成功地用于分离RNA从胰腺具有高完整性2，4，7，10，输液RNA稳定剂与高完整性4，7，10的RNA小鼠胰腺促进隔离的，但是输注解决方案集成到胰腺需要很高的技巧，可能需要专门的工具，如解剖显微镜和手术过程中破坏的组织架构，可能会导致细胞裂解。组织灌注与RNA的稳定剂可能干扰其他应用，包括蛋白分离和组织学染色。此外，这种技术不适合于从人胰腺中分离RNA。其他协议需要由研究者2编制具体的解决方案。
我们报告一个协议来分离RNA从小鼠胰腺诚信度高。钍Ë协议使用先前公布的方法要素，并在很大程度上基于标准的酚/异硫氰酸胍基裂解试剂的协议。它不需要专门的解决方案的准备，也不需要输液剂进入胰腺。对于成功的RNA分离的关键步骤包括一个高酚/胍基裂解试剂对组织的比例，除去相分离和包容核糖核酸酶抑制剂到所得到的RNA溶液先未消化的组织。使用这些和其它的修改，我们通常分离RNA与RIN大于7被用于常规的基因表达分析。
Protocol
Representative Results
Discussion
从被核糖核酸酶丰富的组织中分离RNA代表了分子生物学实验的一大挑战。各种试剂和试剂盒是市售的主要依据RNA提取的酚异硫氰酸胍法。硫氰酸胍变性蛋白，从而减少核糖核酸酶的活性。然而，核糖核酸酶在胰腺的庞大规模需要额外的修改，RNA分离标准协议。协议是在这里报道，基于标准的异硫氰酸胍法还包含几个关键的改进，以及提示RNA的核糖核酸酶丰富的组织如胰腺成功分离。
<p class="jove…Offenlegungen
The authors have nothing to disclose.
Acknowledgements
我们感谢建华岭，雷蒙德·麦克唐纳，高尔文斯威夫特，米歇尔·格里芬，保罗格里波和Satyanarayana Rachagani了他们的宝贵意见，建议和分享他们的协议。这项工作是由授出U01CA111294从俄亥俄州立大学院内研究计划的理念发展奖的支持。
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Power Gen 500 | Fisher Scientific | 14-261-03 | Homogenizer |
| 5424R | Eppendorf | Refrigerated microcentifuge | |
| Trizol reagent | Invitrogen | 15596018 | Lysis reagent |
| Student Vannas spring scissors | Fisher | 91500-09 | use to cut pancreas from attached tissues |
| Surgical scissors, 6 inch | Fisher | 08-951-20 | use to cut scin of mouse |
| Blunt end forceps | Fisher | 1381239 | use to hold pancreas while dissecting |
| Isoflurane USP | Abbott Labs | 4/8/5210 | Anesthetic |
| Rnase out (40 U/µl) | Invitrogen | 10777-019 | Rnase inhbitor |
| Rnase Away | Ambion | 10328-011 | General Rnase inactivator |
| HPLC grade water | Fisher | W5-4 | For washing homogenizer blades |
| Molecular Biology grade water | Hyclone | SH30538.03 | Elution of RNA |
| miRNeasy Mini kit | Qiagen | 217004 | Purification Kit |
| 2 ml microcentrifuge tubes, certified Rnase Dnase free | USA Scientific Plastics | 1620-2700 | |
| 15 ml centrifuge tubes | Falcon | 352099 | |
| 70% ethanol | Fisher | ||
| 100% ethanol | Fisher | ||
| 200 ul pipet tips | Rainin | GPL200F | For cleaning homogenizer blades |
| Chloroform | Fisher | BP1145-1 | |
| Nanodrop | Thermo | ND-1000 | Spectrophotometer |
| Bioanalyzer | Agilent | Capillary electrophoresis analyzer to measue RNA integrity | |
| RNAlater | Ambion | RNA Stabilization Reagent | |
| RNeasy spin columns | Qiagen | spin columns as a part of the miRNeasy Mini kit | |
| Mice | Chales River | strain C57BL/6 | Their age ranged from 4 to 12 months. |
| Mice | Jackson Lab | strain 129 | Their age ranged from 4 to 12 months. |
Referenzen
- Chen, C., Ridzon, D. A., Broomer, A. J., Zhou, Z., Lee, D. H., Nguyen, J. T., Barbisin, M., Xu, N. L., Mahuvakar, V. R., Andersen, M. R., et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 33, e179 (2005).
- Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., Rutter, W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemie. 18, 5294-5299 (1979).
- Chomczynski, P., Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 162, 156-159 (1987).
- Griffin, M., Abu-El-Haija, M., Abu-El-Haija, M., Rokhlina, T., Uc, A. Simplified and versatile method for isolation of high-quality RNA from pancreas. Biotechniques. 52, 332-334 (2012).
- Iyer, V. R., Eisen, M. B., Ross, D. T., Schuler, G., Moore, T., Lee, J. C., Trent, J. M., Staudt, L. M., Hudson Jr, J., Boguski, M. S., et al. The transcriptional program in the response of human fibroblasts to serum. Science. 283, 83-87 (1999).
- Lenstra, J. A., Beintema, J. J. The amino acid sequence of mouse pancreatic ribonuclease. Extremely rapid evolutionary rates of the myomorph rodent ribonucleases. European journal of biochemistry FEBS. 98, 399-408 (1979).
- Mullin, A. E., Soukatcheva, G., Verchere, C. B., Chantler, J. K. Application of in situ ductal perfusion to facilitate isolation of high-quality RNA from mouse pancreas. Biotechniques. 40, 617-621 (2006).
- Rio, D. C., Ares, M., Hannon, G. J., Nilsen, T. W. . RNA A Laboratory Manual. , (2011).
- Streit, S., Michalski, C. W., Erkan, M., Kleeff, J., Friess, H. Northern blot analysis for detection and quantification of RNA in pancreatic cancer cells and tissues. Nat Protoc. 4, 37-43 (2009).
- Zmuda, E. J., Powell, C. A., Hai, T. A method for murine islet isolation and subcapsular kidney transplantation. Journal of Visualized Experiments JoVE. , (2011).
