A method for quantification of absolute amounts of nucleic acids by (RT)-PCR and a new mathematical model for data analysis.
Accurate quantification of nucleic acids by competitive (RT)-PCR requires a valid internal standard, a reference for data normalization and an adequate mathematical model for data analysis. We report here an effective procedure for the generation of homologous RNA internal standards and a strategy for synthesizing and using a reference target RNA in quantification of absolute amounts of nucleic acids. Further, a new mathematical model describing the general kinetic features of competitive PCR was developed. The model extends the validity of quantitative competitive (RT)-PCR beyond the exponential phase. The new method eliminates the errors arising from different amplification efficiencies of the co-amplified sequences and from heteroduplex formation in the system. The high accuracy (relative error <2%) is comparable to the recently developed real time detection 5'-nuclease PCR. Also, corresponding computer software has been devised for practical data analysis.
Vu
H L
HL
Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294-2170, USA. lanvu@uab.edu
Troubetzkoy
S
S
Nguyen
H H
HH
Russell
M W
MW
Mestecky
J
J
D002460:Cell Line; D003627:Data Interpretation, Statistical; D006801:Humans; D007375:Interleukin-1 / Q000235:genetics; D008956:Models, Chemical; D018716:Molecular Mimicry; D009696:Nucleic Acids / Q000032:analysis*; D012313:RNA / Q000032:analysis* / Q000737:chemistry; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012984:Software; D013698:Templates, Genetic
D007375:Interleukin-1; D009696:Nucleic Acids; D012313:RNA