Development of a qRT-PCR assay to determine the relative mRNA expression of two different trypsins in Atlantic cod (Gadus morhua).
A fluorescence resonance energy transfer (FRET) based quantitative RT-PCR method (qRT PCR) was developed in this study for measuring the mRNA expression of trypsins Y and I in the Atlantic cod. Atlantic cod beta-actin was used as the reference gene and standard curves were created for quantification of the mRNA expression levels. For yet unknown reasons, the Atlantic cod (Gadus morhua) produces several trypsins with different characteristics. Trypsin I is the most common and best characterized of these but trypsin Y is a recently discovered enzyme. The recombinant form of trypsin Y was found to have unique characteristics relative to trypsin I. The native form of trypsin Y has proven difficult to isolate from the cod and activity assays do not distinguish between the activities of trypsin I and trypsin Y. The results show that trypsin Y mRNA is expressed in a very low copy number relative to that of trypsin I (ratio of 1:1340), which may explain the difficulty of isolating the native form of trypsin Y.
Pálsdóttir
Helga Margrét
HM
Science Institute, University of Iceland, Laeknagardi, Vatnsmýrarvegi 16, Reykjavík, IS-101, Iceland.
Gudmundsdóttir
Agústa
A
D000818:Animals; D029941:Fish Proteins / Q000096:biosynthesis / Q000235:genetics*; D048389:Gadus morhua / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014357:Trypsin / Q000096:biosynthesis / Q000235:genetics*
D029941:Fish Proteins; D012333:RNA, Messenger; D014357:Trypsin