Objective： Laser capture microdisection has become indispensable to the analysis of the difference of gene expression between human bladder transitional cell and bladder transitional cell carcinoma （BTCC）. However,...Objective： Laser capture microdisection has become indispensable to the analysis of the difference of gene expression between human bladder transitional cell and bladder transitional cell carcinoma （BTCC）. However, to obtain sufficient RNA from laser-capture microdissected cells is quite difficult. The study was designed to determinc a feasible technical routine to isolate transitional cells from bladder membrane, separate carcinoma cclls from stromal cells and to amplify the RNA isolated from laser-capture microdissected cells. Methods： Bladder transitional cell were obtained from frozen sections of bladder membrane applying LCM, by the same token, BTCC cells from frozen sections of BTCC tissue. Then RNA was extracted and linearly amplified in vitro. The expression levels of β-actin in primary total RNA and amplified RNA were detected using RT-PCR. Results： That RNA integrity was good after LCM was confirmed by control experiment Ⅰ; By control experiment Ⅱ, the correlation between the number of LCM-shooting and RNA quantity undcr arranged conditions was preliminarily confirmed. About 0.5-2.5kb RNA fragments were obtained after RNA amplification and β-actin levels were integral. Conclusion： Laser capture microdissection combined with RNA linear amplification in vitro can be successfully applied to obtain pure objective cells for research. The integrity of the amplified RNA is good and can be employed in further research.展开更多
Objective：To isolate ceils of cardiac conduction system （CCS） with laser capture microdissec tion （LCM） and extract and evaluate quality of small amount of RNA from ceils of CCS. Methods： Cryo star sections were...Objective：To isolate ceils of cardiac conduction system （CCS） with laser capture microdissec tion （LCM） and extract and evaluate quality of small amount of RNA from ceils of CCS. Methods： Cryo star sections were followed by H-E staining. 20 pieces of H-E stained eryostat sections were scraped and its RNA was assessed to insure that RNA didn＇t degrade in dyeing and dehydration process. Ceils of CCS were captured with LCM and quality of small amount of RNA was verified with RT-PCR. Results： Ceils of CCS isolated with LCM had clear morphology after staining. High quality RNA was extracted from LCM samples and scraped tissues; 18S rRNA and 28S rRNA were seen distinctly on gel eleetrophoresis. Low level of small amount of RNA extracted from LCM sample was below the limit of detection on gel eleetrophoresis or ultraviolet speetrophotometer. The housekeeping genes β-aetin and GAPDH were successfully amplified with small amount of RNA. Conclusion ：This study resolves the problem of acquiring material of CCS precisely that hinders gene research of CCS. It is found out that the method is easy and reliable to extract and assess the quality of small amount of RNA from mierodisseeted ceils of CCS.展开更多
文摘Objective： Laser capture microdisection has become indispensable to the analysis of the difference of gene expression between human bladder transitional cell and bladder transitional cell carcinoma （BTCC）. However, to obtain sufficient RNA from laser-capture microdissected cells is quite difficult. The study was designed to determinc a feasible technical routine to isolate transitional cells from bladder membrane, separate carcinoma cclls from stromal cells and to amplify the RNA isolated from laser-capture microdissected cells. Methods： Bladder transitional cell were obtained from frozen sections of bladder membrane applying LCM, by the same token, BTCC cells from frozen sections of BTCC tissue. Then RNA was extracted and linearly amplified in vitro. The expression levels of β-actin in primary total RNA and amplified RNA were detected using RT-PCR. Results： That RNA integrity was good after LCM was confirmed by control experiment Ⅰ; By control experiment Ⅱ, the correlation between the number of LCM-shooting and RNA quantity undcr arranged conditions was preliminarily confirmed. About 0.5-2.5kb RNA fragments were obtained after RNA amplification and β-actin levels were integral. Conclusion： Laser capture microdissection combined with RNA linear amplification in vitro can be successfully applied to obtain pure objective cells for research. The integrity of the amplified RNA is good and can be employed in further research.
文摘Objective：To isolate ceils of cardiac conduction system （CCS） with laser capture microdissec tion （LCM） and extract and evaluate quality of small amount of RNA from ceils of CCS. Methods： Cryo star sections were followed by H-E staining. 20 pieces of H-E stained eryostat sections were scraped and its RNA was assessed to insure that RNA didn＇t degrade in dyeing and dehydration process. Ceils of CCS were captured with LCM and quality of small amount of RNA was verified with RT-PCR. Results： Ceils of CCS isolated with LCM had clear morphology after staining. High quality RNA was extracted from LCM samples and scraped tissues; 18S rRNA and 28S rRNA were seen distinctly on gel eleetrophoresis. Low level of small amount of RNA extracted from LCM sample was below the limit of detection on gel eleetrophoresis or ultraviolet speetrophotometer. The housekeeping genes β-aetin and GAPDH were successfully amplified with small amount of RNA. Conclusion ：This study resolves the problem of acquiring material of CCS precisely that hinders gene research of CCS. It is found out that the method is easy and reliable to extract and assess the quality of small amount of RNA from mierodisseeted ceils of CCS.
