Multiplex relative RT-PCR method for verification of differential gene expression.

Differential display, suppression subtractive hybridization and other techniques for identification of differentially expressed genes produce fragments of cDNA from mRNAs whose differences in abundance must be verified. This report describes a relative multiplex RT-PCR assay that facilitates the analysis of large numbers of samples for differences in mRNA abundance without the use of radioactivity or blotting. The species of interest is co-amplified with 18S rRNA over a range of cycles followed by electrophoresis through ethidium bromide-agarose gels. Intensities of the bands of interest, normalized for 18S band intensities, are plotted as a function of cycle number. Regression equations fitted to the curves are used to calculate the number of cycles necessary for each sample's normalized signal to reach a threshold intensity. Differences between samples in the number of cycles required to reach that threshold reflect differences in the original abundances of those species. A comparison with results previously obtained using northern blots showed that relative differences as small as 20% and as large as an order of magnitude are accurately detected. The simplicity of the assay allows its routine application in both research and teaching laboratories.

Acclimation of Photosynthesis and Dark Respiration of a Submersed Angiosperm beneath Ice in a Temperate Lake.

Ceratophyllum demersum L. remained physiologically active beneath ice of a southeastern Michigan lake. The effect of seasonally low photosynthetic photon flux density (PPFD) and cold but nonfreezing temperature on whole-plant physiology was studied. Net photosynthesis was measured at six temperatures and 12 PPFDs. Net photosynthesis, soluble protein concentration, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein concentration, and Rubisco activity of winter plants were 32, 31, 33, and 70% lower, respectively, than those of plants collected in the summer. Optimum temperatures for net photosynthesis of winter and summer plants were 5 and 30[deg]C, respectively. Dark respiration of winter plants was up to 313% greater than that of summer plants. Reduced Rubisco activity and increased dark respiration interacted to reduce net photosynthesis. Interaction of reduced net photosynthesis and increased dark respiration increased CO2 and light compensation points and the light saturation point of winter plants. Growth of C. demersum was limited by the ambient phosphorus concentration of lake water during summer. Apical stem segments of winter-collected plants had 54 and 35% more phosphorus and nitrogen, respectively, than summer-collected plants. Physiologically active perennation beneath ice enabled C. demersum to accumulate phosphorus during the winter when it was most abundant. Partial uncoupling of phosphorus acquisition from utilization may reduce phosphorus limitation upon growth during the summer when phosphorus concentration is seasonally the lowest.