Abstract
In order to identify the optimal internal control for relative real-time PCR when studying target gene expression in the red alga Porphyra yezoensis, we quantified the expression of seven housekeeping genes (18S ribosomal RNA, 30S ribosomal protein S8, Polyubiquitin-2, Glyceraldehyde-3-phosphate dehydrogenase, Elongation factor 1-alpha, Beta-tubulin and Actin 3) at different life-history stages. Absolute quantification was done by normalization to total RNA quantity and by normalization to genomic DNA quantity. We used these two normalization approaches, comparing the differences of expression levels of all candidate housekeeping genes between any two generations and across three life-history stages (filamentous sporophytes, leafy gametophytes and conchospores). We found GAPDH had the best stability in all cases and we recommend that GAPDH be considered as a potential internal control for gene expression studies at different life-history stages in P. yezoensis.
Highlights
Porphyra (Bangiales, Rhodophyta) includes more than 100 species distributed worldwide [1]
P. yezoensis culture The filamentous sporophytes were cultured in sterile seawater with added nutrients with constant aeration at 20uC with 50ummol of photons m22 s21 (12 h light/12 h dark)
In the melting curve analysis, the amplicons of all seven genes revealed a single product with a melting temperature in accord with the expected value (Figure 3)
Summary
Porphyra (Bangiales, Rhodophyta) includes more than 100 species distributed worldwide [1]. The multiphasic life history of Porphyra can be observed in the laboratory within a few months [10], accelerating research on growth, reproduction and photosynthetic mechanisms [11,12,13]. These characteristics have prompted increasing attention to Porphyra, which has been proposed as a model system for physiological and genetic investigation [14,15]. The red alga Porphyra yezoensis is one of the most extensively cultivated species in Japan, China and South Korea. It has a complex heteromorphic life cycle with an alternation between macroscopic haploid leafy gametophytes and microscopic filamentous sporophytes. One question that needs to be answered is whether expression levels of housekeeping genes are constant in the various life-history stages?
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