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Info. Vol.4 - No.4 (2010.12.20)
Title Evaluation and application of RNAs derived from laser microdissected specimens using DNA microarray for expression genomics
Authors Jung Woo Eun1, Ho-Sang Jeong2, Eun-Jung Kim2, Young Min Ahn3, Won Sang Park1, Jung Young Lee1 & Suk Woo Nam1
Institutions 1Department of Pathology, Microdissection Genomics Research Center, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea
2Department of Toxicological Research, National Institute of Toxicological Research, Korea Food & Drug Administration, 194 Tongil-ro, Eunpyeong-gu, Seoul 122-704, Korea
3Department of Kidney System, College of Oriental Medicine, Kyung Hee University, Seoul 122-704, Korea
Correspondence and requests for materials should be addressed to S.W. Nam ( swnam@catholic.ac.kr)
Abstract Laser capture microdissection (LCM) technique has been widely applied to isolate a homogeneous population of cells from heterogeneous tissues. Combined use of microdissection and large-scale DNA microarrays is a powerful tool to research global transcriptional landscape of the clinical specimens. However, the scope of performing microarray using intact RNA derived from LCM isolated cells is very limited due to the instability of mRNA during the LCM process. To overcome this issue, we estimated limitation of the amount of RNA and optimized the RNA extraction procedure for LCM-derived RNA to reproduce RNA with consistent quality for DNA chip application. The three different RNA extraction methods, and amplification methods for LCM-derived RNAs were compared for better quality of hybridization performance of DNA chips. The Trizol reagent method produced RNA of better quality and yield in the LCMderived samples. However, amplification of LCMderived RNA did not fully compensate for the RNA content of amplified RNA (cRNA) derived from the direct dissection of tissue samples. In addition, hybridization performance of LCM-derived cRNAs was not better than that of direct dissection-derived cRNAs, without using LCM. For the reproducibility of data, the correlation coefficients of the samples derived from the two methods were similar, but reproducibility between the two methods was not highly significant (R=0.762). Taken together, our results suggest that application of LCM-derived RNAs to DNA microarrays may be limited in the quantity or quality of cRNA, and may generate less hybridization potential in DNA microarray experiments, but this laser-based microdissection method can be applied to conduct more precise and exquisite assessment of gene expression analysis in heterogeneous tissue samples.
Keyword Laser capture microdissection (LCM), RNA extraction, RNA quality, cRNA amplification, DNA microarray
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