Quantitation of mRNA expression in glomeruli using laser-manipulated microdissection and laser pressure catapulting

被引:27
作者
Nagasawa, Y [1 ]
Takenaka, M [1 ]
Matsuoka, Y [1 ]
Imai, E [1 ]
Hori, M [1 ]
机构
[1] Osaka Univ, Div Nephrol, Dept Internal Med & Therapeut, Grad Sch Med A8, Suita, Osaka 5600871, Japan
关键词
laser beam; TGF-beta; real time PCR; anti-Thy1.1; glomerulonephritis; cell microdissection;
D O I
10.1046/j.1523-1755.2000.00894.x
中图分类号
R5 [内科学]; R69 [泌尿科学(泌尿生殖系疾病)];
学科分类号
1002 ; 100201 ;
摘要
Background. Laser-manipulated microdissection (LMM) is a method to cut out a single cell or limited tiny region from a specimen under microscopic observation by a laser beam. Laser pressure catapulting (LPC) is a method to push up and collect samples that were microdissected using a strong laser. Methods. To induce experimental glomerulonephritis, anti-Thy1.1 monoclonal antibody (OX-7) was injected intrave nously into rats. Control and disease model kidneys were obtained. Six-micrometer thick cryostat sections were mounted onto a 1.35 mu m thin polyethylene membrane. Ten glomeruli were collected from 6 mu m frozen sections of rat kidney by LMM and LPC. Isolated glomeruli were used to quantitate the expression of mRNA by real-time polymerase chain reaction (PCR). Results. Transforming growth factor-beta 1 (TGF-beta 1) mRNA was not detected in glomeruli isolated by the LMM and the LPC methods on day 0, although G3PDH mRNA was measurable in the same samples. On day 7 after the treatment with OX-7, the ratio of TGF-beta 1/G3PDH mRNA was 1.89 +/- 0.96 (N = 6). Conclusions. We established methods to isolate glomeruli from standard histochemical specimens by LMM and LPC, and to quantify mRNA expression in the targeted glomeruli using real-time PCR. We confirmed the up-regulation of TGF-beta 1 mRNA expression in isolated glomeruli from frozen sections of the anti-Thy1.1 glomerulonephritis model.
引用
收藏
页码:717 / 723
页数:7
相关论文
共 36 条
  • [1] Inhibition of TGF-beta 1 expression by antisense oligonucleotides suppressed extracellular matrix accumulation in experimental glomerulonephritis
    Akagi, Y
    Isaka, Y
    Arai, M
    Kaneko, T
    Takenaka, M
    Moriyama, T
    Kaneda, Y
    Ando, A
    Orita, Y
    Kamada, T
    Ueda, N
    Imai, E
    [J]. KIDNEY INTERNATIONAL, 1996, 50 (01) : 148 - 155
  • [2] Laser-assisted preparation of single cells from stained histological slides for gene analysis
    Becker, I
    Becker, KF
    Rohl, MH
    Hofler, H
    [J]. HISTOCHEMISTRY AND CELL BIOLOGY, 1997, 108 (4-5) : 447 - 451
  • [3] Becker I, 1996, LAB INVEST, V75, P801
  • [4] BECKER-ANDRE M, 1991, Methods in Molecular and Cellular Biology, V2, P189
  • [5] Cell sampling - Laser capture microdissection: Molecular analysis of tissue
    Bonner, RF
    EmmertBuck, M
    Cole, K
    Pohida, T
    Chuaqui, R
    Goldstein, S
    Liotta, LA
    [J]. SCIENCE, 1997, 278 (5342) : 1481 - &
  • [6] TRANSFORMING GROWTH FACTOR-BETA-1 INDUCES EXTRACELLULAR-MATRIX FORMATION IN GLOMERULONEPHRITIS
    BORDER, WA
    RUOSLAHTI, E
    [J]. CELL DIFFERENTIATION AND DEVELOPMENT, 1990, 32 (03): : 425 - 432
  • [7] SUPPRESSION OF EXPERIMENTAL GLOMERULONEPHRITIS BY ANTISERUM AGAINST TRANSFORMING GROWTH FACTOR-BETA-1
    BORDER, WA
    OKUDA, S
    LANGUINO, LR
    SPORN, MB
    RUOSLAHTI, E
    [J]. NATURE, 1990, 346 (6282) : 371 - 374
  • [8] BORDER WA, 1994, NEW ENGL J MED, V331, P1286
  • [9] PREPARATION AND STUDY OF FRAGMENTS OF SINGLE RABBIT NEPHRONS
    BURG, M
    GRANTHAM, J
    ABRAMOW, M
    ORLOFF, J
    [J]. AMERICAN JOURNAL OF PHYSIOLOGY, 1966, 210 (06): : 1293 - &
  • [10] Comparison of histologic stains for use in PCR analysis of microdissected, paraffin-embedded tissues
    Burton, MP
    Schneider, BG
    Brown, R
    Escamilla-Ponce, N
    Gulley, ML
    [J]. BIOTECHNIQUES, 1998, 24 (01) : 86 - +