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Reference genes for normalizing transcription in diploid and tetraploid Arabidopsis.

Wang H, Wang J, Jiang J, Chen S, Guan Z, Liao Y, Chen F - Sci Rep (2014)

Bottom Line: However, conservation was less good among the tetraploid accessions, with the transcription of seven of the 21 genes being undetectable in all allotetraploids.The most stable gene was AT5G46630, while AT1G13440 was the unstable one.Hence, the choice of reference gene(s) for A. thaliana is quite wide, but with respect to the analysis of transcriptomic data derived from the tetraploids, it is probably necessary to select more than one reference gene.

View Article: PubMed Central - PubMed

Affiliation: 1] College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China [2] Jiangsu Province Engineering Lab for Modern Facility Agriculture Technology &Equipment, Nanjing 210095, China.

ABSTRACT
Published transcription data from a set of 19 diploid Arabidopsis thaliana and 5 tetraploid (3 allo- and 2 auto- tetraploid) Arabidopsis accessions were re-analysed to identify reliable reference genes for normalization purposes. Five conventional and 16 novel reference genes previously derived from microarray data covering a wide range of abundance in absolute expression levels in diploid A. thaliana Col-0 were employed. Transcript abundance was well conserved for all 21 potential reference genes in the diploid A. thaliana accessions, with geNorm and NormFinder analysis indicating that AT5G46630, AT1G13320, AT4G26410, AT5G60390 and AT5G08290 were the most stable. However, conservation was less good among the tetraploid accessions, with the transcription of seven of the 21 genes being undetectable in all allotetraploids. The most stable gene was AT5G46630, while AT1G13440 was the unstable one. Hence, the choice of reference gene(s) for A. thaliana is quite wide, but with respect to the analysis of transcriptomic data derived from the tetraploids, it is probably necessary to select more than one reference gene.

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Average expression stability values (M) and the pairwise variation (V) metric calculated by geNorm.(a) M values of the 21 reference genes among 19 diploid A. thaliana accessions. A low value of M indicates greater stability. The seven columns shown in black refer to genes not transcribed in at least one tetraploid accession. (b) The pairwise variation (V) metric calculated to indicate the optimal number of reference genes required for normalization. A decrease in V indicates that the addition of the gene in question should improve normalization accuracy.
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f2: Average expression stability values (M) and the pairwise variation (V) metric calculated by geNorm.(a) M values of the 21 reference genes among 19 diploid A. thaliana accessions. A low value of M indicates greater stability. The seven columns shown in black refer to genes not transcribed in at least one tetraploid accession. (b) The pairwise variation (V) metric calculated to indicate the optimal number of reference genes required for normalization. A decrease in V indicates that the addition of the gene in question should improve normalization accuracy.

Mentions: Stability was assessed based on geNorm and NormFinder software. The former associates a stability value (M) with each potential reference gene, where a low M reflects stability and a high M instability5. An M value of < 0.5 is conventionally accepted for a reference, but with high M value (≥0.5) should be avoided11. NormFinder ranks genes according to the similarity of their transcript abundances, applying a model-based approach6. The geNorm analysis showed that the M value of 19 out of the 21 candidate genes was < 0.5, suggesting that any of one (or more) of them was appropriate as a reference sequence(s). The most stable sequences were AT5G46630 (encoding a clathrin adaptor complex subunit), AT1G13320 (PP2A subunit), AT4G26410 (uncharacterised conserved protein), AT5G60390 (EF-1α) and AT5G08290 (mitosis protein YLS8) (Fig. 2a). NormFinder analysis resulted in a similar set of stable genes: AT1G13320 and AT5G46630 emerged as the most stable (Table 2). Nevertheless, even these reference genes could be unstable in two special samples, for example, the RKPM of AT1G13320 was 1.3 times of minimum between diploid WU and TSU samples, while that of AT4G34270, which was identified by both geNorm and NormFinder as being a relatively unstable sequence had the closest transcript abundance (RKPM(WU/TSU) = 14.59/14.06). The pairwise variation (V) metric generated by geNorm was informative with respect to determining the optimal number of reference genes necessary for accurate normalization. A Vn/Vn + 1 value of < 0.15 indicates that an additional reference gene is not required51213. Across the 19 diploid accessions, this metric was below the threshold for the inclusion of a third reference sequence for each initial pair chosen (Fig. 2b).


Reference genes for normalizing transcription in diploid and tetraploid Arabidopsis.

Wang H, Wang J, Jiang J, Chen S, Guan Z, Liao Y, Chen F - Sci Rep (2014)

Average expression stability values (M) and the pairwise variation (V) metric calculated by geNorm.(a) M values of the 21 reference genes among 19 diploid A. thaliana accessions. A low value of M indicates greater stability. The seven columns shown in black refer to genes not transcribed in at least one tetraploid accession. (b) The pairwise variation (V) metric calculated to indicate the optimal number of reference genes required for normalization. A decrease in V indicates that the addition of the gene in question should improve normalization accuracy.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4209459&req=5

f2: Average expression stability values (M) and the pairwise variation (V) metric calculated by geNorm.(a) M values of the 21 reference genes among 19 diploid A. thaliana accessions. A low value of M indicates greater stability. The seven columns shown in black refer to genes not transcribed in at least one tetraploid accession. (b) The pairwise variation (V) metric calculated to indicate the optimal number of reference genes required for normalization. A decrease in V indicates that the addition of the gene in question should improve normalization accuracy.
Mentions: Stability was assessed based on geNorm and NormFinder software. The former associates a stability value (M) with each potential reference gene, where a low M reflects stability and a high M instability5. An M value of < 0.5 is conventionally accepted for a reference, but with high M value (≥0.5) should be avoided11. NormFinder ranks genes according to the similarity of their transcript abundances, applying a model-based approach6. The geNorm analysis showed that the M value of 19 out of the 21 candidate genes was < 0.5, suggesting that any of one (or more) of them was appropriate as a reference sequence(s). The most stable sequences were AT5G46630 (encoding a clathrin adaptor complex subunit), AT1G13320 (PP2A subunit), AT4G26410 (uncharacterised conserved protein), AT5G60390 (EF-1α) and AT5G08290 (mitosis protein YLS8) (Fig. 2a). NormFinder analysis resulted in a similar set of stable genes: AT1G13320 and AT5G46630 emerged as the most stable (Table 2). Nevertheless, even these reference genes could be unstable in two special samples, for example, the RKPM of AT1G13320 was 1.3 times of minimum between diploid WU and TSU samples, while that of AT4G34270, which was identified by both geNorm and NormFinder as being a relatively unstable sequence had the closest transcript abundance (RKPM(WU/TSU) = 14.59/14.06). The pairwise variation (V) metric generated by geNorm was informative with respect to determining the optimal number of reference genes necessary for accurate normalization. A Vn/Vn + 1 value of < 0.15 indicates that an additional reference gene is not required51213. Across the 19 diploid accessions, this metric was below the threshold for the inclusion of a third reference sequence for each initial pair chosen (Fig. 2b).

Bottom Line: However, conservation was less good among the tetraploid accessions, with the transcription of seven of the 21 genes being undetectable in all allotetraploids.The most stable gene was AT5G46630, while AT1G13440 was the unstable one.Hence, the choice of reference gene(s) for A. thaliana is quite wide, but with respect to the analysis of transcriptomic data derived from the tetraploids, it is probably necessary to select more than one reference gene.

View Article: PubMed Central - PubMed

Affiliation: 1] College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China [2] Jiangsu Province Engineering Lab for Modern Facility Agriculture Technology &Equipment, Nanjing 210095, China.

ABSTRACT
Published transcription data from a set of 19 diploid Arabidopsis thaliana and 5 tetraploid (3 allo- and 2 auto- tetraploid) Arabidopsis accessions were re-analysed to identify reliable reference genes for normalization purposes. Five conventional and 16 novel reference genes previously derived from microarray data covering a wide range of abundance in absolute expression levels in diploid A. thaliana Col-0 were employed. Transcript abundance was well conserved for all 21 potential reference genes in the diploid A. thaliana accessions, with geNorm and NormFinder analysis indicating that AT5G46630, AT1G13320, AT4G26410, AT5G60390 and AT5G08290 were the most stable. However, conservation was less good among the tetraploid accessions, with the transcription of seven of the 21 genes being undetectable in all allotetraploids. The most stable gene was AT5G46630, while AT1G13440 was the unstable one. Hence, the choice of reference gene(s) for A. thaliana is quite wide, but with respect to the analysis of transcriptomic data derived from the tetraploids, it is probably necessary to select more than one reference gene.

Show MeSH