Related Experiment Videos
Detection of complement protein mRNA in human astrocytes by the polymerase chain reaction
D L Gordon1, V M Avery, D L Adrian
1Department of Microbiology and Infectious Diseases, Flinders Medical Centre, Bedford Park, Australia.
Abstract:
Primer sets specific for complement proteins, C3, factor B and factor I, were designed and used to amplify cDNA from cultured human astrocyte mRNA by the polymerase chain reaction. Appropriately sized PCR products of 506 bp, 885 bp and 146 bp, respectively, were generated and specificity was confirmed with Southern blotting using an enhanced chemiluminescence detection system. The sensitivity of detection was high, with amplified product from cDNA of approximately 6250 cells readily visualized. C3 and factor B have previously been reported to be produced by murine astrocytes; however, this is the first report indicating synthesis of C3, factor B and factor I by human astrocytes. These results indicate that PCR is a simple and sensitive technique to detect mRNA transcripts for proteins of the alternative pathway of complement in human astrocytes.
Insights
Human astrocytes synthesize complement proteins C3, factor B, and factor I. This study demonstrates the polymerase chain reaction (PCR) as a sensitive method for detecting these crucial immune molecules in astrocytes.
Area of Science:
- Neuroscience
- Immunology
- Molecular Biology
Background:
- The alternative pathway of complement is a key component of the innate immune system.
- Astrocytes play roles in central nervous system immunity.
- Previous studies indicated complement protein production by murine astrocytes.
Purpose of the Study:
- To investigate the synthesis of complement proteins C3, factor B, and factor I by human astrocytes.
- To establish a sensitive method for detecting mRNA transcripts of these proteins in human astrocytes.
Main Methods:
- Design of specific primer sets for complement proteins C3, factor B, and factor I.
- Amplification of complementary DNA (cDNA) from cultured human astrocyte messenger RNA (mRNA) using polymerase chain reaction (PCR).
- Confirmation of PCR product specificity via Southern blotting with enhanced chemiluminescence detection.
Main Results:
- Specific PCR products of expected sizes (506 bp, 885 bp, 146 bp) were generated for C3, factor B, and factor I.
- Southern blotting confirmed the specificity of the amplified products.
- The PCR method demonstrated high sensitivity, visualizing amplified product from approximately 6250 cells.
Conclusions:
- This is the first report demonstrating the synthesis of complement proteins C3, factor B, and factor I by human astrocytes.
- Polymerase chain reaction (PCR) is confirmed as a simple and highly sensitive technique for detecting these complement protein mRNA transcripts in human astrocytes.
- Findings suggest human astrocytes actively participate in the local immune response within the central nervous system via complement production.