Quantitative analysis of transforming growth factor beta isoforms mRNA in the human corneal epithelium.
TGF-beta is an important mediator of cell growth, differentiation, and proliferation and plays a significant role in both normal and pathological corneal tissue. However, the quantitative relations between TGF-beta1, -beta2 and -beta3 isoforms in human cornea still remain unclear. Therefore, the aim of this study was to determine the gene expression profile of TGF-betas in order to evaluate quantitative relations between the examined transcripts in human corneal epithelium. Transcriptional activity of TGF-beta1, 2, 3, GAPDH and beta-actin genes was estimated on the basis of mRNA copy number per 1 microg of total RNA using the real-time QRT-PCR technique with the SYBR Green I chemistry. Specificity of RT-PCR reaction was confirmed by determination of the characteristic melting temperature for each amplimer. Additionally, the RT-PCR products were separated on 6% polyacrylamide gels and visualized with silver salts. Expression of all TGF-beta genes for the corneal epithelium was determined. Comparable analysis of mRNA copies/1 mug of total RNA for each TGF-beta isoform showed that: TGF-beta1 > TGF-beta2; TGF-beta3 > TGF-beta2; TGF-beta1 = TGF-beta3 (ANOVA test P < 0.0001; post-hoc Tukey's test: TGF-beta1 and TGF-beta2, P = 0.0306; TGF-beta3 and TGF-beta2, P = 0.0045; TGF-beta1 and TGF-beta3 NS). We found different expression of the TGF-beta1, -2 and -3 isoforms in the human corneal epithelium. Such differential expression of TGF-betas suggests that each of them may play a specific role in corneal tissue.
Strzalka
B
B
Department of Molecular Biology, Medical University of Silesia, Sosnowiec, Poland. bstrzalka@slam.katowice.pl
Dorecká
M
M
Stanik-Walentek
A
A
Kowalczyk
M
M
Kapral
M
M
Romaniuk
W
W
Mazurek
U
U
Swiatkowska
L
L
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000328:Adult; D019573:Epithelium, Corneal / Q000378:metabolism*; D005260:Female; D020869:Gene Expression Profiling; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D008875:Middle Aged; D020033:Protein Isoforms / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism
D000199:Actins; D020033:Protein Isoforms; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases