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Another important difference between the novel reference genes identified by our analysis and more traditional controls are their absolute expression levels. While EF-1α , UBQ10 , and GAPDH usually belong to the first percentile of highly expressed genes in Arabidopsis, the novel genes shown in Figure 1B have expression levels at least 10- to 20-fold lower (Supplemental Fig. 6). Other genes covering an extended range of mean expression levels can be found in Supplemental Table I.

Complete expression profiles, including all available experimental series, for the genes shown in Tara worn out jeans Clearance Ebay Buy Cheap New Marketable Online Lowest Price For Sale Cheap Supply WvRL1
, for At5g09810 ( ACT7 , formerly called ACT2 ), two other reference genes (i.e. At5g44200 / CBP20 ; At5g25760 / UBC ) qPCR primers that are marketed by Sigma-Aldrich, and for several other genes selected from Supplemental Table I are presented in Supplemental Figure 6. These genes were selected because they appeared on Top100 lists or extended Top500 lists of two or more experimental series (see selection criteria in Supplemental Table II) and because most displayed much lower absolute expression on ATH1 arrays than traditional reference genes ( Tiered Fringe Shorts TOV Best Authentic Clearance Limited Edition Get New Sale For Cheap Free Shipping Explore 7Rhm7Ee
). Overall selection was biased toward genes that are stably expressed in the developmental series because this series is the most complex one, as mentioned above.

View this table:
Table II.

Arabidopsis Gene IDs of traditional reference genes are in bold. % P Calls, Percentage of Present calls within the experimental series, as attributed by MAS5 software.

Vandesompele et al. (2002) developed an algorithm named geNORM that determines the expression stability of reference genes. The calculated gene-stability measure ( M ) relies on the principle that the expression ratio of two ideal internal reference genes is identical in all samples, regardless of the experimental condition or cell type. Accordingly, variation of the expression ratios of two reference genes shows that one or both genes are not stably expressed, hence M > 0. Within a set of reference genes, expression stability for each gene can thus be determined by pair-wise comparison with all other reference genes across all experimental conditions and subsequent calculation of the geometrically averaged sd of log-transformed reference gene ratios.

We used this method as an alternative approach to validate and rank the traditional and novel reference genes, using ATH1 data from the developmental series ( 2018 Cheap Sale Yummie Shaping Thong Manchester For Sale MG4wlOU
; Supplemental Fig. 6). The average expression stability value ( M ) for most traditional genes (black bars in California Dreams Dress SUPERFOXX Cheap Sale Footlocker Pictures Get To Buy Sale For Sale Big Discount For Sale Cheap Price From China j1F87isWj
) was considerably higher (i.e. expression is less stable) than for most of the novel reference genes identified by our approach (white bars). Once again, the polyubiquitin genes At5g25760 ( UBC ) and At4g05320 ( UBQ10 ) showed the most stable expression within the subset of traditional genes with their M values being comparable to those of most of the novel reference genes ( Fig. 2 ). The novel gene with the highest M value, At1g62930 , was chosen because it was represented in three Top100 lists (see Supplemental Table II), although none of these were related to development. Hence, it is not really surprising that it performed less well than other genes as a reference for normalization of developmental expression data. There was good agreement between the average expression stability ( M ) value and the calculated CV value for this set of genes: Linear regression analysis yielded a correlation coefficient ( R 2 ) of 0.91. When the geNORM algorithm was used on combined ATH1 data from all experimental series (Supplemental Fig. 7), the picture remained the same: Novel genes generally had a lower M value, i.e. more stable expression, than traditional genes, with the exception of UBQ10 ( At4g05320 ).

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