Meeting Abstract
Bats have a high reliance on the passive, paracellular absorption of glucose. This contrasts with non-flying mammals that rely extensively on glucose transporters. This could derive from among-species differences in the permeability of the tight junctions across which paracellular absorption occurs. Tight junctions are complex structures composed of many proteins (particularly claudins), and the protein makeup of the tight junctions is thought to affect their permeability characteristics. We desired to use qPCR to investigate the pattern of claudin gene expression and how that pattern varies between bats and non-flying mammals, but could find no general protocol for comparing qPCR data across species. We will discuss the particular problems associated with making cross-species comparisons, including finding comparable normalization genes, differences in the efficiencies of reactions, and variable fluorescence of SYBR green with differing amplicons. Our solutions to these problems included: use of a functionally relevant reference gene, designing primers that work with multiple species when possible, accurate measurement of reaction efficiency for every amplicon, and controlling for variation in fluorescence via normalizing by AT content (adenine/thymine content) of the amplicon. We will also apply these methods to intestinal tight junction proteins in several bat and non-flying rodent species to understand how they are associated with differences in paracellular nutrient absorption. Supported by USA NSF and Argentina CONICET.
