O SlideShare utiliza cookies para otimizar a funcionalidade e o desempenho do site, assim como para apresentar publicidade mais relevante aos nossos usuários. Se você continuar a navegar o site, você aceita o uso de cookies. Leia nosso Contrato do Usuário e nossa Política de Privacidade.
O SlideShare utiliza cookies para otimizar a funcionalidade e o desempenho do site, assim como para apresentar publicidade mais relevante aos nossos usuários. Se você continuar a utilizar o site, você aceita o uso de cookies. Leia nossa Política de Privacidade e nosso Contrato do Usuário para obter mais detalhes.
AMPLIFICATION OF THREE OSTEOBLAST GENE
MARKERS USING REVERSE TRANSCRIPTASE-PCR
NURUL FATIHAH BINTI MOHAMMED AZANAN
DR. AZLINA AHMAD
ASSITED BY: EN. FUAD YUSOF
Stem cells in human exfoliated deciduous teeth (SHED)
Immature, unspecialized cells in the human deciduous
teeth that are able to grow into specialized cell types by a
process known as “differentiation”.
Contains multipotent stem cells and were identified to
highly proliferate and capable of differentiating into a
variety of cell types including osteoblast (bone cells),
adipocytes (fat cells), neural cells and odonblast (Miura et
Multipotent stem cell has potential as cell sources for bone
regeneration (Chadipiralla et al.,2010).
Human exfoliated teeth (SHED)
Figure 1: Human exfoliated teeth (SHED) structure
Osteoblast-specific transcription factor that were studied:
Bone morphogenetic protein-2 (BMP2) is responsible for
mineralized tissue formation (Yang et al., 2009).
Osteopontin (OPN) is involved in the remodeling of the
bone tissue where it promotes adhesion of the bone cells
to the bone surface (Wejheden, 2006).
Runt related gene2 (RUNX2) also known as core binding
factor a1 (CBFA1) is an important gene in osteoblast
differentiation and bone formation.
To optimize the amplification of BMP2, OPN and RUNX2
from human stem cell exfoliated deciduous teeth (SHED)
by Reverse Transcriptase-Polymerase Chain Reaction
Gene DNA sequence Melting
55.1˚C 659 bp Khodavirdi et al.,
57.4˚C 222 bp Papathanasiou et
51.9˚C 230 bp Abdallah et al.,
Table 1: PCR primer sequences and their product size
Table 2: Composition of PCR reaction
PCR components Volume (µl)
2X My Taq 1-Step Mix 5 µl
Forward primer 0.4 µl
Reverse primer 0.4 µl
Ribosafe RNase inhibitor (10u/µl) 0.2 µl
DEPC treated water 1.4 µl
Reverse transcriptase 0.1 µl
RNA sample 2.5 µl
Total volume 10 ul
Table 3: Standard RT-PCR conditions
PCR steps Annealing
Annealing Time Cycle
Initial Denaturation 95˚C 1 min
95˚C 10 sec
Annealing X 10 sec
Extension 72˚C 30 sec
• Optimization of BMP2 and OPN:
Figure 2: PCR products of BMP2 and OPN visualized by
1% of agarose gel electrophoresis using 6 µl sample + 2
µl loading dye showed a clear single band for BMP2 at
222bp but unclear band (faint) for OPN at size 550 bp.
Figure 3: PCR products of BMP2 and OPN visualized by
1% of agarose gel electrophoresis using 6 µl for BMP2
and 8 µl for OPN showed clear bands at size 222bp and
Optimization of RUNX2
Figure 4: First trial PCR optimization of RUNX2 visualized by 1% of agarose gel electrophoresis
using 3 µl sample and 2 µl loading dye showed multiple bands (unspecific) for each annealing
Figure 5: Second trial PCR optimization of RUNX2
visualized by 1% agarose gel electrophoresis using 3 µl
sample and 2 µl loading dye showed single band for
each annealing temperature with the presence of
Figure 6: The volume (1.5 µl samples + 1 loading dye) of
PCR optimization of RUNX2 samples were reduced and
visualized by 1.5% agarose gel electrophoresis. It showed
clear single bands for each temperature except for
temperature 62.6˚C (faint band).
Figure 7: Third trial PCR optimization of RUNX2
visualized by 1% agarose gel electrophoresis using 3 µl
sample and 2 µl loading dye showed single band at
temperature 61.2 ˚C and 63.0˚C but unspecific band for
59.4˚C and 65.1˚C. However no band presented at
Figure 8: The volume (1.5 µl samples + 1 loading
dye) of PCR optimization of RUNX2 samples were
reduced and visualized by 1.5% agarose gel
electrophoresis. It showed clear single bands for
each temperature except for temperature 62.6˚C
Optimum PCR condition:
gel con. %
BMP2 61.9˚C 10
34X 1% 222 bp 222 bp
OPN 63.2˚C 10
35X 1% 659 bp 550 bp
RUNX2 63.6˚C 10
40X 1.5% 230 bp 230 bp
BMP2 and OPN:
Managed to obtain single band at the target size for
each of genes after the loading preparation was
Thus, the loading preparation also influences the
intensity of band
By changing a few parameters: increasing of
annealing temperature and agarose gel percentage
and also by reducing loading preparation, a single
band at target size 230bp managed to obtain.
Optimum annealing temperature of RUNX2 is 63.6˚C
since it showed good band appearance than others
Optimization of BMP2, OPN and RUNX2 from human
stem cell exfoliated deciduous teeth (SHED) by RT-
PCR were successfully to obtain a single target band
after several parameters of PCR were changed.
Chadipiralla, K., Yochim, J. M., Bahuleyan, B., Huang, C. Y. C., Garcia-Godoy, F.,
Murray, P. E., & Stelnicki, E. J. (2010). Osteogenic differentiation of stem
cells derived from human periodontal ligaments and pulp of human
exfoliated deciduous teeth. Cell and tissue research, 340(2),323-333.
Miura, M., Gronthos, S., Zhao, M., Lu, B., Fisher, L. W., Robey, P. G., & Shi, S.
(2003). SHED: stem cells from human exfoliated deciduous
teeth.Proceedings of the National Academy of Sciences, 100(10), 5807-
Wejheden, C., Brunnberg, S., Hanberg, A., & Lind, P. M. (2006). A rapid and
sensitive response to dioxin exposure in the osteoblastic cell line UMR-
106.Biochem. Biophys. Res. Commun, 134(1), 116-120.
Yang, X., Van der Kraan, P. M., Bian, Z., Fan, M., Walboomers, X. F., & Jansen, J.
A. (2009). Mineralized tissue formation by BMP2-transfected pulp stem
cells. Journal of dental research, 88(11), 1020-1025.
• Dr. Sarina Sulong
• Dr. Tan Huay Lin
• Dr. Azlina Ahmad
• Prof Dr. Ravindran Ankathil
• Dr. Teguh Haryo Sasongko
• Science Officers
• Cytogenetics and Molecular staff
• Dental of Sciences Staff & Craniofacial Lab
• Students and all the members of Human Genome