A Simple Approach for Evaluating Total MicroRNA Extraction from Mouse Brain Tissues
Author(s)
Jack G. Walleshauser III,
Trace Kessler,
Danielle Morse,
Bakhos A. Tannous,
Norman H. L. Chiu*
Affiliation(s)
Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, USA. Molecular Neuro- genetics Unit, Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, USA. Department of Nanoscience, Joint School of Nanoscience and Nanoengineering, Greensboro, USA.
Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, USA. Molecular Neuro- genetics Unit, Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, USA. Department of Nanoscience, Joint School of Nanoscience and Nanoengineering, Greensboro, USA.
ABSTRACT
For the analysis of microRNA, a common approach is to first extract microRNA from cellular samples prior to any specific microRNA detection. Thus, it is important to determine the quality and yield of extracted microRNA. In this study, solid-phase extraction was used to isolate small RNA (<200 nt), which included microRNA, from mouse brain tissues. By using standard UV absorbance measurements, the amount of small RNA in the extracted RNA samples was determined. To determine the presence of microRNA, each RNA sample was analyzed by PAGE with SYBR? Green II staining. Testing for contamination of any small DNA fragments, RNase and cellular peptides or proteins were systematically carried out. By scanning the gel image obtained from PAGE analysis, the average percentage of total microRNA (19 - 25 nt) in the extracted RNA samples was determined to be equal to 2.3 ± 0.5%. The yield of total microRNA was calculated to be ~0.5ng of microRNA per milligram of frozen mouse brain tissue. In comparison to other methods that require the use of expensive specialized instrumentation, the approach of combining the standard UV absorbance and PAGE analysis represents a simple and viable method for evaluating the quality and yield of microRNA extraction from tissue samples.
For the analysis of microRNA, a common approach is to first extract microRNA from cellular samples prior to any specific microRNA detection. Thus, it is important to determine the quality and yield of extracted microRNA. In this study, solid-phase extraction was used to isolate small RNA (<200 nt), which included microRNA, from mouse brain tissues. By using standard UV absorbance measurements, the amount of small RNA in the extracted RNA samples was determined. To determine the presence of microRNA, each RNA sample was analyzed by PAGE with SYBR? Green II staining. Testing for contamination of any small DNA fragments, RNase and cellular peptides or proteins were systematically carried out. By scanning the gel image obtained from PAGE analysis, the average percentage of total microRNA (19 - 25 nt) in the extracted RNA samples was determined to be equal to 2.3 ± 0.5%. The yield of total microRNA was calculated to be ~0.5ng of microRNA per milligram of frozen mouse brain tissue. In comparison to other methods that require the use of expensive specialized instrumentation, the approach of combining the standard UV absorbance and PAGE analysis represents a simple and viable method for evaluating the quality and yield of microRNA extraction from tissue samples.
Cite this paper
J. G. Walleshauser III, T. Kessler, D. Morse, B. A. Tannous and N. H. L. Chiu, "A Simple Approach for Evaluating Total MicroRNA Extraction from Mouse Brain Tissues," Journal of Analytical Sciences, Methods and Instrumentation, Vol. 2 No. 1, 2012, pp. 5-12. doi: 10.4236/jasmi.2012.21002.
J. G. Walleshauser III, T. Kessler, D. Morse, B. A. Tannous and N. H. L. Chiu, "A Simple Approach for Evaluating Total MicroRNA Extraction from Mouse Brain Tissues," Journal of Analytical Sciences, Methods and Instrumentation, Vol. 2 No. 1, 2012, pp. 5-12. doi: 10.4236/jasmi.2012.21002.
References
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[1] [1] A. Fire, S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver and C. C. Mello, “Potent and Specific Genetic Interference by Double-Stranded RNA in Caenorhabditis Elegan,” Nature, Vol. 391, 1998, pp. 806-811. doi:10.1038/35888 [2] R. K. Slotkin and R. Martienssen, “Transposable Elements and the Epigenetic Regulation of the Genome,” Nature Reviews Genetics, Vol. 8, No. 4, 2007, pp. 272- 285. doi:10.1038/nrg2072 [3] C. D. Novina and P. A. Sharp, “The RNAi Revolution,” Nature, Vol. 430, 2004, pp. 161-164. doi:10.1038/430161a [4] W. Filipowicz, S. N. Bhattacharyya and N. Sonenberg, “Mechanisms of Post-Transcriptional Regulation by mic- roRNAs: Are the Answers in Sight?” Nature Reviews Genetics, Vol. 9, No. 2, 2008, pp. 102-114. doi:10.1038/nrg2290 [5] M. S. Nicoloso and G. A. Calin, “MicroRNA Involvement in Brain Tumors: From Bench to Bedside,” Brain Pathology, Vol. 18, No. 1, 2008, pp. 122-129. doi:10.1111/j.1750-3639.2007.00119.x [6] W. Kong, J. Zhao, L. He and J. Q. Cheng, “Strategies for Profiling MicroRNA Expression,” Journal of Cellular Physiology, Vol. 218, No. 1, 2008, pp. 22-25. doi:10.1002/jcp.21577 [7] A. Aravin and T. Tuschl, “Identification and Characterization of Small RNAs Involved in RNA Silencing,” FEBS Letters, Vol. 579, No. 26, 2005, pp. 5830-5840. doi:10.1016/j.febslet.2005.08.009 [8] P. Chomczynski and N. Sacchi, “Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Ph- enol-Chloroform Extraction,” Analytical Biochemistry, Vol. 162, No. 1, 1987, pp. 156-159. doi:10.1016/0003-2697(87)90021-2 [9] A. M. Krichevsky, K. S. King, C. P. Donahue, K. Khrapko and K. S. Kosik, “A MicroRNA Array Reveals Extensive Regulation of MicroRNAs during Brain Development,” RNA, Vol. 9, 2003, pp. 1274-1281. doi:10.1261/rna.5980303 [10] G. Sun, H. Li and J. J. Rossi, “Cloning and Detecting Signature MicroRNAs from Mammalian Cells,” Methods in Enzymology, Vol. 427, 2007, pp. 123-138. doi:10.1016/S0076-6879(07)27007-7 [11] T. Watanabe, Y. Totoki, H. Sasaki. N. Minami and H. Imai, “Analysis of Small RNA Profiles during Development,” Methods in Enzymology, Vol. 427, 2007, pp. 155-169. doi:10.1016/S0076-6879(07)27009-0 [12] A. M. Krichevsky, “MicroRNA Profiling: From Dark Matter to White Matter, or Identifying New Players in Neurobiology,” Scientific World Journal, Vol. 7, 2007, pp. 155-166. doi:10.1100/tsw.2007.201 [13] W. Li and K. Ruan, “MicroRNA Detection by Mic-roarray,” Analytical and Bioanalytical Chemistry, Vol. 394, No. 4, 2009, pp. 1117-1124. doi:10.1007/s00216-008-2570-2 [14] M. Hafner, P. Landgraf, J. Ludwig, et al., “Identification of MicroRNAs and Other Small Regulatory RNAs Using cDNA Library Sequencing,” Methods, Vol. 44, No. 1, 2008, pp. 3-12. doi:10.1016/j.ymeth.2007.09.009 [15] S. Griffiths-Jones, K. Saini, et al., “miRBase: Tools for MicroRNA Genomics,” Nucleic Acids Research, Vol. 32, 2008, pp. D154-D158. [16] E. A. Hunt, A. M. Goulding and S. K. Deo, “Direct Detection and Quantification of MicroRNAs,” Analytical Biochemistry, Vol. 387, No. 1, 2009, pp. 1-12. doi:10.1016/j.ab.2009.01.011 [17] E. Varallyay, J. Burhyan and Z. Havelda, “Detection of microRNAs by Northern Blot Analyses Using LNA Probes,” Methods, Vol. 43, No. 2, 2007, pp. 140-145. doi:10.1016/j.ymeth.2007.04.004 [18] K. A. Cissell, S. Shrestha and S. K. Deo, “microRNA Detection: Challenges for the Analytical Chemist,” Anal. Chem, Vol. 79, No. 13, 2007, pp. 4754-4761. doi:10.1021/ac0719305 [19] C. Chen, D. A. Ridzon, A. J. Broomer, et al., “Real-Time Quantification of microRNAs by Stem-Loop RT-PCR,” Nucleic Acids Research, Vol. 33, 2005, pp. e179:1- e179:9. [20] J. Chen, J. Lozach, et al., “Highly Sensitive and Specific microRNA Expression Profiling Using BeadArray Technology,” Nucleic Acids Research, Vol. 36, 2008, pp. e87:1-e87:9. [21] T. W?rdinger, B. A. Tannous, et al., “MiR-296 Regulates Growth Factor Receptor Overexpression in Angiogenic Endothelial Cells,” Cancer Cell, Vol. 14, No. 5, 2008, pp. 382-393. doi:10.1016/j.ccr.2008.10.005 [22] N. C. Mishra, “Nucleases: Molecular Biology and Applications,” John Wiley & Sons, Hoboken, 2002. [23] J. Sambrook and D. W. Russell, “Molecular Cloning: A Laboratory Manual,” 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001. [24] V. V. Didenko, “DNA Probes Using Fluorescence Resonance Energy Transfer (FRET): Designs and Applications,” Biotechniques, Vol. 31, 2001, pp. 1106-1121. [25] Instructional Manual for mirVanaTM miRNA Isolation Kit, Ambion, 2010. http://www.ambion.com/techlib/prot/fm_1560.pdf