Ranking of candidate reference genes according to the BestKeeper analysis of their expression stability.

<p>For dataset symbols, refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192343#pone.0192343.t003" target="_blank">Table 3</a>.</p

Sample datasets for statistical analysis of expression stability of candidate reference genes in <i>A</i>. <i>fatua</i>.

<p>Symbols of sample groups refer to S1 Table. For each sample set, a count (N) of sample combinations included in analysis are given.</p

Pairwise variation (V) analyses of the candidate reference genes.

<p>The pairwise variation (V_n/n+1) was analyzed for the normalization factors NFn and NFn+1 by the geNorm program to determine the optimal number of reference genes for accurate normalization. The cutoff value was proposed to be 0.15, below which the inclusion of an additional reference gene is not necessary. For dataset symbols, refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192343#pone.0192343.t003" target="_blank">Table 3</a>.</p

Relative expression of <i>AfVP1</i> in ND versus PD <i>A</i>. <i>fatua</i> caryopses during incubation on water at 20°C (NPC sample group).

<p>Different normalization factors were used, optimal according to geNorm (A), NormFinder (B, C), BestKeeper (D) and ΔCt method (D). The fold changes indicate the expression levels of <i>AfVP1</i> gene in non-dormant caryopses relative to calibrator (PD caryopses) with assumed value of 1 at each time-point.</p

Reference gene selection for molecular studies of dormancy in wild oat (<i>Avena fatua</i> L.) caryopses by RT-qPCR method

<div><p>Molecular studies of primary and secondary dormancy in <i>Avena fatua</i> L., a serious weed of cereal and other crops, are intended to reveal the species-specific details of underlying molecular mechanisms which in turn may be useable in weed management. Among others, quantitative real-time PCR (RT-qPCR) data of comparative gene expression analysis may give some insight into the involvement of particular wild oat genes in dormancy release, maintenance or induction by unfavorable conditions. To assure obtaining biologically significant results using this method, the expression stability of selected candidate reference genes in different data subsets was evaluated using four statistical algorithms i.e. geNorm, NormFinder, Best Keeper and ΔCt method. Although some discrepancies in their ranking outputs were noticed, evidently two ubiquitin-conjugating enzyme homologs, <i>AfUBC1</i> and <i>AfUBC2</i>, as well as one homolog of glyceraldehyde 3-phosphate dehydrogenase <i>AfGAPDH1</i> and TATA-binding protein <i>AfTBP2</i> appeared as more stably expressed than <i>AfEF1a</i> (translation elongation factor 1α), <i>AfGAPDH2</i> or the least stable α-tubulin homolog <i>AfTUA1</i> in caryopses and seedlings of <i>A</i>. <i>fatua</i>. Gene expression analysis of a dormancy-related wild oat transcription factor <i>VIVIPAROUS1</i> (<i>AfVP1</i>) allowed for a validation of candidate reference genes performance. Based on the obtained results it can be recommended that the normalization factor calculated as a geometric mean of Cq values of <i>AfUBC1</i>, <i>AfUBC2</i> and <i>AfGAPDH1</i> would be optimal for RT-qPCR results normalization in the experiments comprising <i>A</i>. <i>fatua</i> caryopses of different dormancy status.</p></div

Gene expression stability and ranking of seven candidate reference genes based on geNorm algorithm.

<p>Each panel refers to a distinct dataset described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192343#pone.0192343.t003" target="_blank">Table 3</a>. Average stability measure (M) was calculated following stepwise exclusion of the least stable gene. Lower M values indicate more stable gene expression.</p

Candidate genes expression stability value (M) of single and best combination of two genes estimated by NormFinder algorithm in different datasets.

<p>For dataset symbols, refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192343#pone.0192343.t003" target="_blank">Table 3</a>.</p

Gene-specific primers and amplicon characteristics of candidate reference genes and a target gene used in RT-qPCR analysis.

<p>Gene-specific primers and amplicon characteristics of candidate reference genes and a target gene used in RT-qPCR analysis.</p