Detection of complement protein mRNA in human astrocytes by the polymerase chain reaction.
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.
Gordon
D L
DL
Department of Microbiology and Infectious Diseases, Flinders Medical Centre, Bedford Park, Australia.
Avery
V M
VM
Adrian
D L
DL
Sadlon
T A
TA
D001253:Astrocytes / Q000737:chemistry*; D001483:Base Sequence; D015139:Blotting, Southern; D003176:Complement C3 / Q000096:biosynthesis; D011415:Complement Factor B / Q000096:biosynthesis; D003165:Complement System Proteins / Q000032:analysis / Q000096:biosynthesis*; D004247:DNA / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D005340:Fibrinogen / Q000096:biosynthesis; D005455:Fluorescent Antibody Technique; D005784:Gene Amplification; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000032:analysis; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000302:isolation & purification
D003176:Complement C3; D009841:Oligonucleotides; D012333:RNA, Messenger; D005340:Fibrinogen; D003165:Complement System Proteins; D004247:DNA; D011415:Complement Factor B