RNA Isolation fra Mouse Pancreas: en ribonuklease-rige væv
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
Isolering af RNA med høj integritet er nødvendig for rutinemæssige molekylærbiologiske eksperimenter såsom Northern blotting 9, qRT-PCR 1 eller genekspression profilering 5.. De fleste moderne fremgangsmåder til RNA-isolering er baseret på modifikationer af guanidinthiocyanat protokoller 2, 3. Guanidinthiocyanat er en stærk proteindenatureringsmiddel og effektivt vil forstyrre aktiviteten af ribonuclease under de fleste forhold. Den populære Chomczynski og Sacchi 3 kombineret phenol til guanidinthiocyanat lyseopløsning reducere isolation tid til omkring 4 timer. Mange kommercielt tilgængelige RNA-ekstraktionsmetoder reagenser baseret på Chomczynski og Sacchi metode 3.
Ribonuklease rige væv, såsom human eller mus bugspytkirtlen er en yderligere udfordring for at isolere RNA. Mus bugspytkirtlen kan indeholde op til 75 mg ribonuclease 6 hvoraf nogle vil blive frigivet During afbrydelse af pancreasvæv resulterer i væv autolyse. Modifikationer af guanidinthiocyanat protokoller er blevet anvendt til at isolere RNA fra bugspytkirtel med høj integritet 2, 4, 7, 10. Infusion af RNA-stabilisering reagens i muse pancreas lettere isolering af RNA med høj integritet 4, 7, 10, men infusion af løsninger i bugspytkirtlen kræver stor dygtighed, kan kræve specialiserede instrumenter såsom et dissektionsmikroskop og forstyrrer vævsarkitekturen under proceduren, kan resultere i cellelysis. Perfusion væv med RNA stabilisering reagens kan forstyrre andre programmer, herunder protein isolation og histologisk farvning. Endvidere er denne teknik ikke egnet til isolering af RNA fra human pancreas. Andre protokoller kræver udarbejdelse af konkrete løsninger af investigator 2.
Vi rapporterer en protokol til at isolere RNA med høj integritet fra mus bugspytkirtlen. The protokollen bruger elementer af tidligere publicerede metoder og er i vid udstrækning baseret på standarden phenol / guanidinthiocyanat-baserede lysis reagens protokoller. Det kræver ikke udarbejdelse af specialiserede løsninger kræver heller ikke infusion af reagenser i bugspytkirtlen. Kritiske trin til vellykket RNA isolation omfatter en høj phenol / guanidin-baserede lysis reagens væv forholdet, fjernelse af ufordøjet væv forud for fase separation og optagelse af et ribonucleaseinhibitor til den resulterende RNA-løsning. Ved hjælp af disse og andre modifikationer, vi typisk isolere RNA med RIN større end 7, der skal anvendes til rutinemæssig genekspressionsanalyse.
Protocol
Representative Results
Discussion
Isolerende RNA fra væv, som ribonukleaseaktivitet rige repræsenterer en stor udfordring for molekylærbiologiske eksperimenter. Forskellige reagenser og kits er kommercielt tilgængelige, der primært er baseret på phenol guanidinthiocyanat metode til RNA-ekstraktion. Guanidinthiocyanat denaturerer protein og dermed reducerer aktiviteten af ribonuclease. , Selve omfanget af ribonuklease i bugspytkirtlen kræver imidlertid yderligere ændringer standardprotokoller RNA isolation. En protokol er rapporteret her, d…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Vi takker Jianhua Ling, Raymond MacDonald, Galvin Swift, Michelle Griffin, Paul Grippo og Satyanarayana Rachagani for deres nyttige kommentarer, forslag og deling af deres protokoller. Dette arbejde blev støttet af tilskud U01CA111294 og en Idéudvikling Award fra Ohio State University Intramural Research Program.
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. |
References
- 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. Biochimie. 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).
