AMPLIFICATION OF THREE OSTEOBLAST GENE
MARKERS USING REVERSE TRANSCRIPTASE-PCR
(RT-PCR): OPTIMIZATION
PRESENTED BY:
NURUL FATIHAH BINTI MOHAMMED AZANAN
SUPERVISED BY:
DR. AZLINA AHMAD
ASSITED BY: EN. FUAD YUSOF

INTRODUCTION
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
al., 2003).
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.

OBJECTIVE
To optimize the amplification of BMP2, OPN and RUNX2
from human stem cell exfoliated deciduous teeth (SHED)
by Reverse Transcriptase-Polymerase Chain Reaction
(RT-PCR)

METHODOLOGY
Cell culture
RNA extraction
PCR mastermix (Bioline kit)
RT-PCR
Agarose gel electrophoresis

Gene DNA sequence Melting
temperature
(Tm)
Annealing
Temperature
Product
size (bp)
Accoding to
OPN 5’-CATCTCAGAAGCAGAATCTCCTA-3’F
5’-TGATTGATAGTCAGGAACTTTCC-3’R
56.0˚C
54.2˚C
55.1˚C 659 bp Khodavirdi et al.,
2006
BMP2 5’-CCCAGCGTGAAAAGAGAGAC-3’F
5’-CTTCTAGCGTTGCTGCTTCC-3’R
57.4˚C
57.4˚C
57.4˚C 222 bp Papathanasiou et
al., 2012
RUNX2 5’-TCTTCACAAATCCTCCCC-3’F
5’-TGGATTAAAAGGACTTGGTG-3’R
52.6˚C
51.3˚C
51.9˚C 230 bp Abdallah et al.,
2005
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
Temperature (˚C)
Annealing Time Cycle
Initial Denaturation 95˚C 1 min
40 cycles
Denaturation
95˚C 10 sec
Annealing X 10 sec
Extension 72˚C 30 sec

RESULT
• 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
550bp respectively
222bp
200bp
555bp 555bp
222bp

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
temperature
230bp

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
smear
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).
230bp
230bp

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
67.9˚C temperature.
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
(faint band).
230bp
230bp

Optimum PCR condition:
Gene Annealing
temperature
Annealing
time
Cycle Agarose
gel con. %
Expected
PCR size
(bp)
PCR
product
size
(bp)
BMP2 61.9˚C 10
seconds
34X 1% 222 bp 222 bp
OPN 63.2˚C 10
seconds
35X 1% 659 bp 550 bp
RUNX2 63.6˚C 10
seconds
40X 1.5% 230 bp 230 bp

DISCUSSION
BMP2 and OPN:
Managed to obtain single band at the target size for
each of genes after the loading preparation was
increased
Thus, the loading preparation also influences the
intensity of band

RUNX2:
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

CONCLUSION
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.

REFERENCES
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-
5812.
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.

ACKNOWLEDGEMENT
• 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
Centre