[Development of new software (GiMlet) to analyze real-time PCR data based on TaqMan Chemistry and examination of factors affecting variance of the Ct values].
The primary data obtained from real-time PCR is the fluorescence intensity. We developed new software, GiMlet, to analyze the fluorescence data more effectively. To examine factors affecting the variance of the Ct values calculated from the fluorescence data, we conducted measurements of the same test solutions containing defined numbers of copies of the targeted DNA sequence using three kinds of real-time PCR equipment, namely, ABI PRISM 7500, 7700 and 7900HT, under consistent conditions, and analyzed the obtained fluorescence data using GiMlet. We also introduced a new baseline correction algorithm in GiMlet and examined the effect of this correction on the measurement results. The results revealed that the variances of the measured values and the PCR efficiency differed among the three kinds of real-time PCR equipment used and also according to the well-position of the PCR plate. The fluorescence data could be analyzed more precisely using the new algorithm.
Watanabe
Takahiro
T
National Institute of Health Sciences, Tokyo, Japan.
Matsuda
Rieko
R
D000704:Analysis of Variance; D003199:Computer Systems; D005163:Factor Analysis, Statistical; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity; D012984:Software; D012985:Software Design; D013223:Statistics as Topic / Q000379:methods*; D019914:Taq Polymerase
D019914:Taq Polymerase