China Biotechnology, 2005, 25 (3) :74 78

Μέγεθος: px

Εμφάνιση ξεκινά από τη σελίδα:

Download "China Biotechnology, 2005, 25 (3) :74 78"

Ἀρίσταρχος Μοσχοβάκης
5 χρόνια πριν
Προβολές:

1 China Biotechnology, 2005, 25 (3) : ( ) ( ) : (LCM) LCM, RNA : LCM, RNA, RNA RT2 PCR Total RNA 2actin : LCM RNA ; LCM shooting RNA 25 shootings ; 20 shootings RT2PCR 2actin : LCM, RNA, RT2PCR RNA, RNA, RNA, ( laser : : com. tw capture microdissection,lcm) [1 ] g,, LCM,, RNA,, 1 111,, G2, T2 5ml, (1) - 80, - 30 ( Zeiss,HM505E ), OCT(,SAKURA), 10 m [2,3 ],,, 6, OCT - 80 (2) 6 ( h ;, [4,5 ] ),,

2005,25 (3) : 75 RNAsy mini kit Total RNA, I (3) [2,3,6,7 ], g, 2 : RNAlater (Qiagen, RNAlater RNA Stabilizition Reagent) 30 l, 4min,DEPC 5s, 1min, DEPC 5s, (011 %) 10s,DEPC 5s,70 % 15s,95 % 20s,

2 2005,25 (3) : 75 RNAsy mini kit Total RNA, I (3) [2,3,6,7 ], g, 2 : RNAlater (Qiagen, RNAlater RNA Stabilizition Reagent) 30 l, 4min,DEPC 5s, 1min, DEPC 5s, (011 %) 10s,DEPC 5s,70 % 15s,95 % 20s, 25s, 1min,, 113 PixCell II CapSure LCM Macrocaps (Arcturus Engineering, Inc. ), [6 9 ] : Pulse Duration 118ms ;Beam Diameter 15 m ;Beam Power 70mW 30min, RNA [10 ] shootings, 4 shootings, RNA 114 RNA RNeasy Mini Kit ( Qiagen Inc ) RLT Buffer Cap 350 l RLT Buffer 7, 50 l, RNA 015ml eppendorf Cap,, 5min,, (10s), Cap RNA, - 80 RNA RLT Buffer eppendorf min, QIAgredder homogenizer (Qiagen Inc), RNase2free DNase ( the RNase2free Dnase Set,Qiagen Inc) DNA RNA RNeasy Mini Kit 115 RNA Rneasy MinElute Cleanup Kit RNA 116 RNA RT2RCR RNA RT2RCR Promega, 1 A :,40 ;B :,40 ;C:Cpa,40 ;D :Cap,200 Fig. 1 Bladder transitional cell

211 1A B :, ; C Cap, D LCM 2 :, 2 A :,10 20 ;B :,10 20 Fig.

3 76 China Biotechnology Vol. 25 No [11 ], 2actin : 5 ACTG GGAC GACA TGGA GAAG 3 ;5 AGTT TGAT GGAT GCCA CAGG 3,600bp A B :, ; C Cap, D LCM 2 :, 2 A :,10 20 ;B :,10 20 Fig. 2 Bladder transitional cell carcinoma 212 RNA, LCM,RNA 3 :28s 18s, 2 1,,RNA,, RNA 600bp 2actin, 4 3 Total RNA Fig. 3 Control experimentr I total RNA 213 RNA 30 l, 2, 50 l Eppendorf Biophotometer ( Ger Brinkmann Instruments Inc) A 260 A 280 A 260Π ,RNA 485ng 214 RT2PCR RT2PCR 2actin RNA 3 4 2actin 1 :DNA marker ; 2 : 2actin Fig. 4 2actin expression,

4 2005,25 (3) : 77,,,,,,,,,, [12,13 ] LCM,, [14,15 ], LCM,,, ( 1 2) : (1), (2) 4h LCM,,, 30 4h 8 10 LCM (3) RNAlater 70 %, RNA (4) DEPC 5min, LCM (5) LCM,,,, (6) LCM 30min (7) Cap RLT buffer 0. 5ml eppendorf 5min Cap Cap, Cap, Cap ;,, Cap ; : ; ; LCM ;, ;, : Total RNA ( 3) : Total RNA, RNA ; 2actin RNA RT2PCR, 2actin ( 4) RNA, mrna RNA LCM RNA,! [ 1 ] Emmert2Buck M R,Bonner R F,Smith P D,et al. Laser capture microdissection. Science,1996,274 (8) :998 [ 2 ] Lo Y L, Shen C Y. Laser capture microdissection in carcinoma analysis. Methods2Enzymol,2002,356 : [ 3 ] Fend F,SpechtK,Kremer M,et al.laser capture microdissection in pathology. Methods2Enzymol,2002,356 : [ 4 ] Betsuyaku T, Robert M. Laser capture microdissectionand mrna characterization of mouse airway epithelium methodological considerations. Micron,2004, (35 : [ 5 ] Parlato R,Rosica A,Cuccurullo V,et al. A preservation method that allows recovery of intact RNA form tissure dissected by laser capture microdissection. Analytical Biochemistry,2002,300 :

5 78 China Biotechnology Vol. 25 No [ 6 ] Hoffmann M,Olson K,Cavender A,et al. Gene expression in a pure population of odontoblasts isolataed by laser2capture microdissection.j Dent Res,2001,80 (11) : [ 7 ] Grant K,Jerome W G. Laser capture microdissection as an aid to ultrastructural analysis. Microsc Microanal,2002,8 : [ 8 ] Suzuki K,Matsui H, Hasumi M,et al. Gene expression profiles in human BPH: utilization of laser2capture microdissection and quantitative real2time PCR. Anticancer Res,2001,21 : [ 9 ] Bonner R F, Emmert2Buck M, Cole K, et al. Laser capture microdissection :molecular analysis of tissue. Science,278 : [ 10 ] Batorfi J,Ye B,Mok S C,et al. Protein profiling of complete mole and normal placenta using proteinchip analysis on laser capture microdissected cells. Gynecologic Oncology,2003,88 : [ 11 ],,,. RNA.,2002,21 (9) : Han W N,Li H, Wang SH,et al. Cancer,2002,21 (9) : [ 12 ],..,2001,24 (2) :61 63 Wang Zh N,Zhang X. Medical Sciences,2001,24 (2) :61 63 [ 13 ],,,.LCM.,2003,1 :40 42 Guo F ZH,Qiu SH L,Xiao X Y,et al. Mordern Equipment,2003, 1 :40 42 [ 14 ],,, ,2003,20 (10) : Man X B, Tang L, Tang Y X, et al. Chin J Exp Surg, 2003, 20 (10) : [ 15 ] Miura K, Bowman E D, Simon R, et al. Laser capture microdissection and microarray expression analysis of lung adenocarcinoma reveals tobacco smoking and prognosis2related molecular profiles. Cancer Res,2002,62 : Application of Laser Capture Microdissection in Research of Relevant Genes in Bladder Cancer HAO Quan 1 XU Yong 2 LI Wen2lu 1 GAO Xing 3 Qiu Zhi2qiang 4 (1 Tianjin Cancer Hospital,Tianjin Medical University Tianjin , China) (2 The Second Affiliated Hospital,Tianjin Medical University Tianjin , China) (3 School of Medicine,Xian Communication University Xi an , China) (4 Tianjin Cancer Hospital,Tianjin Medical University Tianjin , China) Abstract Objective : Laser Capture Microdisection ( LCM) has become indispensable for obtaining the homogeneous objective cells. Transitional cells were isolated from bladder membrane and carcinoma cells were separated from stromal cells by LCM. RNA were extracted,purified and concentrated from laser2capture microdissected cells for further use. Methods : Bladder transitional cells were obtained from bladder tissue, bladder transitional cell carcinoma (BTCC) cells from BTCC tissue by frozen sections and LCM. Then RNA was extracted,purified and concentrated. The expression level of 2actin in total RNA was detected by RT2PCR. Results :RNA integrity is good after LCM confirmed by control experiment ;By control experiment,the correlation between the number of LCM2shooting and RNA quantity under arranged conditions was preliminary conformed. 2actin level was integral. Conclusion : LCM can be applied to successfully obtain purely objective cells for research. The integrity of RNA is good and can be used in further research. carcinoma Key words Laser Capture Microdissection RT2PCR Bladder transitional cell Bladder transitional cell

Σχετικά έγγραφα

IL - 13 /IL - 18 ELISA PCR RT - PCR. IL - 13 IL - 18 mrna. 13 IL - 18 mrna IL - 13 /IL Th1 /Th2

IL - 13 /IL - 18 ELISA PCR RT - PCR. IL - 13 IL - 18 mrna. 13 IL - 18 mrna IL - 13 /IL Th1 /Th2 344 IL - 13 /IL - 18 1 2 1 2 1 2 1 2 1 2 3 1 2 13 18 IL - 13 /IL - 18 10% / OVA /AL OH 3 5% 16 ~ 43 d 44 d ELISA BALF IL - 13 IL - 18 PCR RT - PCR IL - 13 IL - 18 mrna IL - 13 mrna 0. 01 IL - 18 mrna 0.