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대한생식의학회지 제28권 제3호 2010년
Laser Captured Microdissection을 이용한 유전자 발현에 대한 연구 (I): RT-PCR을 위한 난자의 RNA 추출 및 증폭을 위한 최소한도의 확립
차병원 여성의학연구소1, 포천중문의과대학교2
박창은, 박창은1,고정재1,2,차광렬1,2,이경아1,2*
Analysis of the Gene Expression by Laser Captured Microdissection (I):Minimum Conditions Required for the RNA Extraction from Oocytes and Amplification for RT-PCR
Chang Eun Park1, Jung Jae Ko1,2, Kwang Yul Cha1,2, Kyung-Ah Lee1,2*
1Infertility Medical Center, CHA General Hospital, 2College of Medicine, Pochon CHA University Seoul
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Objective: Recently, microdissection of tissue sections has been used increasingly for the isolation of morphologically identified homogeneous cell populations, thus overcoming the obstacle of tissue complexity for the analysis cell-specific expression of macromolecules. The aim of the present study was to establish the minimal conditions required for the RNA extraction and amplification from the cells captured by the laser captured microdissection. Methods: Mouse ovaries were fixed and cut into serial sections (7 μm thickness). Oocytes were captured by laser captured microdissection (LCM) method by using PixCell IITM system. The frozen sections were fixed in 70% ethanol and stained with hematoxylin and eosin, while the paraffin sections were stained with Multiple stain. Sections were dehydrated in graded alcohols followed by xylene and air-dried for 20 min prior to LCM. All reactions were performed in ribonuclease free solutions to prevent RNA degradation. After LCM, total RNA extraction from the captured oocytes was performed using the guanidinium isothiocyanate (GITC) solution, and subsequently evaluated by reverse transcriptase -polymerase chain reaction (RT-PCR) for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). Results: With the frozen sections, detection of the GAPDH mRNA expression in the number of captured 25 oocytes were not repeatable, but the expression was always detectable from 50 oocytes. With 25 oocytes, at least 27 PCR cycles were required, whereas with 50 oocytes, 21 cycles were enough to detect GAPDH expression. Amount of the primary cDNA required for RT-PCR was reduced down to at least 0.25 μl with 50 oocytes, thus the resting 19.75 μl cDNA can be used for the testing other interested gene expression. Tissue-to-slide, tissue-to-tissue forces were very high in the paraffin sections, thus the greater number of cell procurement was required than the frozen sections. Conclusion: We have described a method for analyzing gene expression at the RNA level with the homogeneously microdissected cells from the small amount of tissues with complexity. We found that LCM coupled with RT-PCR could detect housekeeping gene expression in 50 oocytes captured. This technique can be easily applied for the study of gene expression with the small amount of tissues
키워드 : : Laser captured microdissection, Mouse ovary, Gene expression, RT-PCR
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