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Presentation indsutrial training

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Presentation indsutrial training

  1. 1. 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
  2. 2. 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).
  3. 3. Human exfoliated teeth (SHED) Figure 1: Human exfoliated teeth (SHED) structure
  4. 4. 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.
  5. 5. 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)
  6. 6. METHODOLOGY Cell culture RNA extraction PCR mastermix (Bioline kit) RT-PCR Agarose gel electrophoresis
  7. 7. 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
  8. 8. 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
  9. 9. 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
  10. 10. 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
  11. 11. 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
  12. 12. 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
  13. 13. 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
  14. 14. 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
  15. 15. 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
  16. 16. 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
  17. 17. 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.
  18. 18. 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.
  19. 19. 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

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