Presentation indsutrial training

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 osteoblastdifferentiation and bone formation.

OBJECTIVE

To optimize the amplification of BMP2, OPN and RUNX2from 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

Denaturation95˚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

222bp200bp

555bp 555bp

222bp

Optimization of RUNX2

Figure 4: First trial PCR optimization of RUNX2 visualized by 1% of agarose gel electrophoresisusing 3 µl sample and 2 µl loading dye showed multiple bands (unspecific) for each annealing temperature

230bp

Figure 5: Second trial PCR optimization of RUNX2visualized 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 RUNX2visualized 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).

230bp230bp

Optimum PCR condition:

Gene Annealingtemperature

Annealing time

Cycle Agarosegel 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 10seconds

40X 1.5% 230 bp 230 bp

DISCUSSIONBMP2 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