degradation. Lacking any other explanation, it was tentatively concluded that these

truncated UGT sequences were artifacts of the PCR reaction.




 A 0







 & 700











Figure 5-24. 3' RACE for I4.

 A. Inner PCR, B. Additional round of PCR (PCR reaction loaded in duplicate)

 Quality of RNA. The fact that only two UGT isoforms were identified indicated

some problem with the methodology. Catfish, like the phylogenetically related zebrafish,

was expected to have multiple dissimilar UGT isoforms, particularly in liver. The fact

that RACE failed to amplify the partial DNA sequences on several occasions led to the

postulation that the RT reaction had been performed at a temperature that was too low

(420C, even though this is within the normal operating temperatures for the IVMLV-RT

enzyme), resulting in mRNA that was not folded correctly. In addition, it was noted that

the RACE procedure omitted the heating step for 3 min at 70-850C, prior to the actual

reverse transcription, which helps to unfold the RNA. However, the heating step had been