Real-time PCR quantification of human complement C4A and C4B genes.
BACKGROUND
The fourth component of human complement (C4), an essential factor of the innate immunity, is represented as two isoforms (C4A and C4B) in the genome. Although these genes differ only in 5 nucleotides, the encoded C4A and C4B proteins are functionally different. Based on phenotypic determination, unbalanced production of C4A and C4B is associated with several diseases, such as systemic lupus erythematosus, type 1 diabetes, several autoimmune diseases, moreover with higher morbidity and mortality of myocardial infarction and increased susceptibility for bacterial infections. Despite of this major clinical relevance, only low throughput, time and labor intensive methods have been used so far for the quantification of C4A and C4B genes.


RESULTS
A novel quantitative real-time PCR (qPCR) technique was developed for rapid and accurate quantification of the C4A and C4B genes applying a duplex, TaqMan based methodology. The reliable, single-step analysis provides the determination of the copy number of the C4A and C4B genes applying a wide range of DNA template concentration (0.3-300 ng genomic DNA). The developed qPCR was applied to determine C4A and C4B gene dosages in a healthy Hungarian population (N = 118). The obtained data were compared to the results of an earlier study of the same population. Moreover a set of 33 samples were analyzed by two independent methods. No significant difference was observed between the gene dosages determined by the employed techniques demonstrating the reliability of the novel qPCR methodology. A Microsoft Excel worksheet and a DOS executable are also provided for simple and automated evaluation of the measured data.


CONCLUSIONS
This report describes a novel real-time PCR method for single-step quantification of C4A and C4B genes. The developed technique could facilitate studies investigating disease association of different C4 isotypes.
Szilagyi
Agnes
A
Institute of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary. szilagyi@puskin.sote.hu
Blasko
Bernadett
B
Szilassy
Denes
D
Fust
George
G
Sasvari-Szekely
Maria
M
Ronai
Zsolt
Z
D000328:Adult; D015934:Complement C4a / Q000235:genetics*; D015935:Complement C4b / Q000235:genetics*; D003199:Computer Systems; D004247:DNA / Q000097:blood / Q000235:genetics*; D017931:DNA Primers; D018628:Gene Dosage; D006801:Humans; D006814:Hungary; D016133:Polymerase Chain Reaction / Q000379:methods*; D012984:Software; D019914:Taq Polymerase
D017931:DNA Primers; D015934:Complement C4a; D015935:Complement C4b; D004247:DNA; D019914:Taq Polymerase