Meeting Abstract

P1.93  Monday, Jan. 4  Gene expression in fourth and fifth instar Manduca sexta YEOH, A.J.*; LONG, R.A.; GILLEN, C.M.; HARTLAUB, B.A.; Kenyon College; Kenyon College; Kenyon College; Kenyon College gillenc@kenyon.edu

The scaling exponent for metabolic rate in Manduca sexta is greater than expected based on isometric surface area scaling. We examined whether midgut genes involved in absorption and digestion are expressed differently in fifth versus fourth instar larvae. RNA was isolated from middle and posterior midgut and reverse transcribed to cDNA. Real-time PCR was used to quantify expression of two genes, aminopeptidase N (msAPN3) and potassium amino acid cotransporter (KAAT), by the relative quantification method using 18s ribosomal RNA as an internal control. A general linear model for APN and KAAT ΔCt values identified significant instar and tissue effects. The best model provided a good fit, with an R-squared value of 0.9335. In posterior midgut, KAAT expression was 2.5-3.5 fold higher while APN was 1.3-1.4 fold lower in fifth compared to fourth instar larvae. Expression of KAAT was 3.5 fold higher in middle compared to posterior midgut. To evaluate the sensitivity of standard statistical methods to real time PCR data, a Monte Carlo simulation of the coverage probabilities for Ct, ΔCt and ΔΔCt was conducted for the normal, uniform, exponential, t and chi-square distributions using parameter estimates derived from our data. Coverage probabilities for Ct and ΔCt were close to the 95% confidence level, indicating that the interval estimates are relatively insensitive to distributional assumptions. A notable exception was the exponential distribution of Ct, where the coverage probability dropped to 0.88. Coverage probabilities for ΔΔCt were reasonable when using the mean of a comparison group, but not when arbitrarily selecting an individual measurement for comparison. (Supported by NSF-DMS #0827208 and Kenyon College)