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button  Structure-Property Relationships in Dental Polymers and Composites
     button  Nanocomposite Dental Materials
  button  Structure-Property Relationships of Hydrogels for Dental and Craniofacial Applications
  button  The Effect of an Organogelator on Bioactive Dental Composites
  button   High-throughput and combinatorial methods for measuring the mechanical properties of dental materials
button  Combinatorial Methods for Rapid Screening of Biomaterials
  button  High-throughput Method for Determining Young’s Modulus of Polymer Blends
  button  Inflammatory Cytokine Quantification of Cell-SCK Interactions via RT-PCR
  button  Peptide Derivatized SCK Nanoparticles
  button  Real-Time Polymerase Chain Reaction
  button  Gradient Library Screening of Cell-Material Interactions
  button  Surface Energy Gradients for Characterizing Cell-Material Interactions
  button  High-throughput Method for Characterizing Cell Response to Polymer Crystallinity
  button   Cellular Response to Bis-GMA/TEGDMA Vinyl Conversion Gradients
button  Metrologies for Tissue Scaffolds
  button  Focal Adhesions of Osteoblasts on Poly(d,l-lactide)/Poly(vinyl alcohol) Blends by Confocal Fluorescence Microscopy
  button   2D -->3D Cell / Scaffold Interactions
  button  Development of a Reference Scaffold
  button   In Vitro Cartilage Development
  button   Gene Expression Profiles of Cells in Response to Tyrosine Polycarbonate Blends
  button Broadband Coherent Anti-Stokes Raman Scattering (CARS) Microscopic Imaging
  button Collinear Optical Coherence and Confocal Fluorescence Microscopies
 

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Real-Time Polymerase Chain Reaction
 

Introduction
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The molecular interface between the biomaterial and the external surface of the cell is a critical point of interaction for tissue-engineered products. The nature of both the physical and chemical interactions can cause cells to transmit a variety of critical biological signals. In response to external stimuli, cells initiate a genetic response to determine which regulatory pathway the cell will traverse. These pathways include chronic and acute inflammation, differentiation, proliferation, and extracellular matrix production, which ultimately affects the well-being of the cell and determines the success of the tissue-engineered scaffold. We have developed a set of genetic tests based on Quantitative Real Time Polymerase Chain Reaction (QRT-PCR) for assessing complex cellular responses.
 

Experimental Approach

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Experimental Approach
 
Experimental Approach
 
In a typical experiment, mRNA is extracted from a cell population following a defined incubation period with the substrate. Using a reverse transcriptase (RT) enzyme, mRNA is converted to the cDNA template necessary for amplification. Then the cDNA, gene specific primers, a DNA polymerase, and a fluorescent moiety are utilized to amplify and label the amplicon generated. The gene product accumulation is then measured during the exponential phase of the amplification reaction.
 

Inflammatory Response Quantification
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Inflammatory Response Quantification
 
The copy number is obtained by extrapolating to a standard gene curve of known concentration and copy number to yield quantitative data. The assay also includes the analysis of mRNA that does not change in relative abundance (18S), which serves as an internal control.
 

A List of the Gene Specific Markers and their Corresponding Biological Processes

Process Gene Maker
Inflammation I1-1, TNF- ß
Extracellular Fibronectin, Collagen I & II, Actin
Differentiation Aggrecan, Osteopontin, Osteocalcin
 

For More Information:

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Bailey L., Washburn N, Simon C.G., Chan E, Wang F.W. The Quantification of Inflammatory Cellular Responses Using Real-Time Polymerase Chain Reaction (RT-PCR) 2004 Journal of Biomedical Materials Research, 69A(2), 305-313.
 

NIST Contributors

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Matthew L. Becker
Lee Ann O. Bailey
 
 
 
 
 
 
 
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Biomaterials Group
Polymers Division
Materials Science and Engineering Laboratory
 
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