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20071116_mtDNA workshop
Idenfitied Differentially Expressed Genes in
Keratoconus
Ji Eun Lee, Jong Soo Lee
Department of Ophthlamology, Graduate School of Medicine, Pusan National University
20071116_mtDNA workshop
Authors have no financial interest in any materials discussed in this article.
Purpose
• It is valuable to examine the genes of the keratocytes that are involved
in the thinning of the cornea because investigation of the fundamental
pathogenesis is important to understand the disease of the keratoconus.
• The purpose of this study is to evaluate the pathogenesis of keratoconus
through differentially expressed genes in human keratocyte of
keratoconus.
Methods
• Culture of human keratocyte
• mRNA isolation
• cDNA synthesis
• Annealing Control Primer (ACP)-
based GeneFisingTM PCR
• Cloning and Sequencing
• RT-PCR
• Quantitative Real-Time PCR
Figure 1. ACPTM structure. APCTM technology provides a primer with annealing specificity
to the temple and allows only real products to be amplifies, such that it enables the
researchers to find only real products as a result. A: core sequence - annealing at the first
stage of PCR (targeting). B: universal sequence - annealing at the second stage of PCR. C:
regulator- regulating the functions of A and B.
Results
500bp
1000bp1 : 정상각막
2 : 원추각막
A EBA A
1 2
E
1000bp
500bp
1 2
A
1 2
C
1 2
D
1 2
B
1 2 1 2 1 2
F G H
Figure 2. ACP-based PCR product of the eight verified differentially expressed genes
showing increased or decreased levels of expression in keratoconus (arrows). 1: normal
cornea. 2: keratoconus. A: BMP4. B: ACTA 2. C: CFL 1, D: GRCC 10, E: TIMP 3, F:
MRVI1, G: TIMP1, H: SSTR1.
Results
GAPDH
BMP4
1 2
GAPDH
ACTA2
1 2
GAPDH
CFL1
1 2
GAPDH
GRCC10
1 2
GAPDH
TIMP3
1 2
GAPDH
MRVI1
1 2
GAPDH
TIMP1
1 2
GAPDH
SSTR1
1 2
Figure 3. Depicted are RT-PCR products of the eight verified genes and a housekeeping gene
(GAPDH). 1: normal cornea. 2: keratoconus.
Cytoskeleton - CFL1, GRCC10, SSTR1, TIMP1, TIMP3
Wound healing - ACTA2
Apoptosis - BMP4, CFL1, MRVI1
Results
Figure 4. Quantitative real-time polymerase chain reaction. Quantitative real-time polymerase chain reaction of the eight verified genes using GAPDH as endogenous control showed that BMP4 (A), CFL1 (B), MRVI1 (C) were increased by 1.6, 3.3, and 11 fold, respectively, whereas ACTA2 (D), GRCC10 (E), TIMP3 (F), TIMP1 (G), and SSTR1 (H) were decreased by 4.5, 2.7, 14, 8.5, and 1.8 fold, respectively in keratoconus relatively to norma l cornea.
Conclusions
• We found differential expression of genes related to apoptosis, as well as those
related to cytoskeleton structure and wound healing in keratoconus, using
GeneFishingTM PCR.
• This was based on the PCR method using the mRNA of keratocytes in normal
cornea and keratoconus.
• We also confirmed the importance of the apoptosis, cytoskeleton, and wound
healing of keratocytes as an important cause of keratoconus through the
differential expressions of genes.
• We anticipate that gene therapy techniques using such genes will suppress the
progress and side effects of keratoconus in the future.