Simple Modifications to Standard TRIzol® Protocol Allow High-Yield RNA Extraction from Cells on Resorbable Materials

Juliana Tsz Yan Lee, Wai Hung Tsang, King Lau Chow

.

DOI: 10.4236/jbnb.2011.21006                

Abstract

Resorbable bioceramics are attractive for medical applications such as bone substitution. Biochemical analysis on cells cultured on these biomaterials is vital to predict the impact of the materials in vivo and RNA extraction is an essential step in gene expression study using RT-qPCR. In this study, we describe simple modifications to the TRIzol® RNA extraction protocol widely used in biology and these allow high-yield extraction of RNA from cells on resorbable calcium phosphates. Without the modifications, RNA is trapped in the co-precipitated calcium compounds, rendering TRIzol® extraction method infeasible. Among the modifications, the use of extra TRIzol® to dilute the lysate before the RNA precipitation step is critical for extraction of RNA from porous ?-tricalcium phosphate (?-TCP) discs. We also investigate the rationale behind the undesirable precipitation so as to provide clues about the modifications required for other resorbable materials with high application potential in bone tissue engineering.

Keywords

Calcium Phosphate, Resorbable Materials, RNA Extraction, TRIzol, Acid Guanidium Thiocyanate - Phenol - Chloroform Extraction, TRI Reagent, TRIsure

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	L. L. Hench and J. M. Polak, “Third-Generation Biomedical Materials,” Science, Vol. 295, No. 5557, 2002, pp. 1014-1017. doi:10.1126/science.1067404
[2]	M. Vallet-Regi? and J. M. Gonza?lez-Calbet, “Calcium Phosphates as Substitution of Bone Tissues,” Progress in Solid State Chemistry, Vol. 32, No. 1-2, 2004, pp. 1-31. doi:10.1016/j.progsolidstchem.2004.07.001
[3]	P. Habibovic, U. Gbureck, C. J. Doillon, D. C. Bassett, C. A. van Blitterswijk and J. E. Barralet, “Osteoconduction and Osteoinduction of Low-Temperature 3D Printed Bioceramic Implants,” Biomaterials, Vol. 29, No. 7, 2008, pp. 944-953. doi:10.1016/j.biomaterials.2007.10.023
[4]	C. Knabe, A. Houshmand, G. Berger, P. Ducheyne, R. Gildenhaar, I. Kranz and M. Stiller, “Effect of Rapidly Resorbable Bone Substitute Materials on the Temporal Expression of the Osteoblastic Phenotype in vitro,” Journal of Biomedical Materials Research-Part A, Vol. 84, No. 4, 2008, pp. 856-868. doi:10.1002/jbm.a.31383
[5]	P. Chomczynski and N. Sacchi, “Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol- Chloroform Extraction,” Analytical Biochemistry, Vol. 162, No. 1, 1987, pp. 156-159. doi:10.1016/0003-2697(87)90021-2
[6]	P. Chomczynski and N. Sacchi, “The Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction: Twenty-Something Years on,” Nature Protocols, Vol. 1, No. 2, 2006, pp. 581-585. doi:10.1038/nprot.2006.83
[7]	P. Niemeyer, U. Krause, J. Fellenberg, P. Kasten, A. Seckinger, A. D. Ho and H. Simank, “Evaluation of Mineralized Collagen and α-Tricalcium Phosphate as Scaffolds for Tissue Engineering of Bone Using Human Mesenchymal Stem Cells,” Cells Tissues Organs, Vol. 177, No. 2, 2004, pp. 68-78. doi:10.1159/000079182
[8]	U. Mayr-Wohlfart, J. Fiedler, K. Gnther, W. Puhl and S. Kessler, “Proliferation and Differentiation Rates of a Human Osteoblast-Like Cell Line (SaOS-2) in Contact with Different Bone Substitute Materials,” Journal of Biomedical Materials Research, Vol. 57, No. 1, 2001, pp. 132-139. doi:10.1002/1097-4636(200110)57:1<132::AID-JBM1152>3.0.CO;2-K
[9]	M. Y. Deng, H. Wang, G. B. Ward, T. R. Beckham and T. S. McKenna, “Comparison of Six RNA Extraction Methods for the Detection of Classical Swine Fever Virus by Real-Time and Conventional Reverse Transcription-PCR,” Journal of Veterinary Diagnostic Investigation, Vol. 17, No. 6, 2005, pp. 574-578.
[10]	L. Z. Santiago-Va?zquez, L. K. Ranzer and R. G. Kerr, “Comparison of Two Total RNA Extraction Protocols Using the Marine Gorgonian Coral Pseudopterogorgia Elisabethae and its Symbiont Symbiodinium sp.,” Electronic Journal of Biotechnology, Vol. 9, No. 5, 2006, pp. 598-603.
[11]	X. Xiang, D. Qiu, R. D. Hegele and W. C. Tan, “Comparison of Different Methods of total RNA Extraction for Viral Detection in Sputum,” Journal of virological methods, Vol. 94, No. 1-2, 2001, pp. 129-135. doi:10.1016/S0166-0934(01)00284-1
[12]	H. Yuan, Z. Yang, J. D. De Bruijn, K. De Groot and X. Zhang, “Material-Dependent Bone Induction by Calcium Phosphate Ceramics: A 2.5-Year Study in Dog,” Biomaterials, Vol. 22, No. 19, 2001, pp. 2617-2623. doi:10.1016/S0142-9612(00)00450-6
[13]	J. Sambrook and D. W. Russell, “Molecular Cloning: A Laboratory Manual,” 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001.
[14]	M. M. Amiji and B. J. Sandmann, “Applied Physical Pharmacy,” McGraw-Hill, Medical Publishing Division, New York, 2003.
[15]	E. Fern?ndez, F. J. Gil, M. P. Ginebra, F. C. M. Driessens, J. A. Planell and S. M. Best, “Calcium Phosphate Bone Cements for Clinical Applications. Part I: Solution Chemistry,” Journal of Materials Science: Materials in Medicine, Vol. 10, No. 3, 1999, pp. 169-176. doi:10.1023/A:1008937507714
[16]	S. V. Dorozhkin and M. Epple, “Biological and Medical Significance of Calcium Phosphates,” Angewandte Chemie-International Edition, Vol. 41, No. 17, 2002, pp. 3130-3146. doi:10.1002/1521-3773(20020902)41:17<3130::AID-ANIE3130>3.0.CO;2-1
[17]	P. Chomczynski and K. Mackey, “Substitution of Chloroform by Bromo-Chloropropane in the Single-Step Method of RNA Isolation,” Analytical Biochemistry, Vol. 225, No. 1, 1995, pp. 163-164. doi:10.1006/abio.1995.1126