We have tested our in silico designed primers for over 1,000 mouse genes. These genes are known to be expressed in mouse livers by EST library analysis. Therefore we have used mouse liver total RNA in the validation experiments. Real-time PCR melting curve analysis and gel electrophoresis are performed to evaluate real-time PCR success. In most cases, we observe a single peak in the melting curve and a single band on gel for one PCR reaction. Occasionally, the gel and melting curve analyses do not agree with each other. In these cases, we have also performed DNA sequencing to make sure there is only a single PCR product. The design success rate, defined as a single PCR product with reasonable amplification efficiency, was 99% using these validation methods in an initial screen. A somewhat lower rate (approximately 93%) has been observed for a primer collection spanning nearly all of the mouse genome (A. Spandidos, personal communication).
Figure 5.3 shows a validation experiment for 16 cytochrome P-450 genes. Cytochrome P-450 is a large gene family with ~90% sequence similarity between some family members. Despite this very high sequence homology, all 16 P-450 primer pairs lead to single PCR products, validated by both gel electrophoresis and DNA sequencing. The melting curve analyses also indicate single PCR products for most of the PCRs (6 examples shown in Figure 5.3(B)).
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