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Selection of appropriate reference genes for quantitative real-time PCR in Oxytropis ochrocephala Bunge using transcriptome datasets under abiotic stress treatments.

Zhuang H, Fu Y, He W, Wang L, Wei Y - Front Plant Sci (2015)

Bottom Line: To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable reference genes is required.When normalized with the most stable reference genes, the expression patterns of the three target genes were in accordance with those in the transcriptome data, indicating that the reference genes selected in this study are suitable.The usage of inappropriate reference gene would cause misinterpretation.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Resource Biology and Biotechnology in Western China, Department of Life Science, Northwest University Xi'an, China.

ABSTRACT

Background: Oxytropis ochrocephala Bunge, an indigenous locoweed species in China, poses great threats to livestock on grasslands. There is a need for further genetic study in the plants per se, for understanding the basis of its acclimation mechanism in various unfavorable environmental conditions and to implement effective control measures. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method for gene expression analysis. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable reference genes is required. The aim of this study was to select the most stable reference genes for transcriptional analysis in O. ochrocephala.

Results: We selected 12 candidate reference genes, 18S ribosomal RNA (18S RNA), actin2/7 (ACT7), β-actin (ACTB), actin101 (ACT101), actin11 (ACT11), β-tubulin (TUB), α-tubulin (TUA), glyceraldehyde-3-phosphate dehydrogenase-1 (GAPDH1), GAPDH2, metallothionein-like protein (MET), fructose-bisphosphate aldolase (FBA) and histone H3 (HIS), from the transcriptome datasets of O. ochrocephala and determined the suitability by analyzing their expression levels when exposed to a range of abiotic stress conditions. By employing software packages including geNorm, NormFinder and BestKeeper, HIS, ACT7, and ACT101 were assessed as the most suitable set for normalization in all samples. When normalized with the most stable reference genes, the expression patterns of the three target genes were in accordance with those in the transcriptome data, indicating that the reference genes selected in this study are suitable.

Conclusions: The study provided appropriate reference genes for accurate normalization in qRT-PCR analysis in O. ochrocephala and emphasized the importance of validating reference genes for gene expression analysis under specific experimental condition. The usage of inappropriate reference gene would cause misinterpretation.

No MeSH data available.


Related in: MedlinePlus

Pairwise variation (V) of 12 candidate reference genes calculated by geNorm to determine the optimal number of reference genes for accurate normalization. Asterisk indicates the optimal number of reference genes required for normalization.
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Figure 3: Pairwise variation (V) of 12 candidate reference genes calculated by geNorm to determine the optimal number of reference genes for accurate normalization. Asterisk indicates the optimal number of reference genes required for normalization.

Mentions: The optimal number of the reference genes required for accurate normalization was determined by pairwise variation (Vn/Vn+1). In the subsets of drought and cold stress, the V2/3 value was below 0.15 (0.144 and 0.107, respectively), which suggested that two reference genes should be used for normalization. In the salt treatment subset, three reference genes were sufficient for accurate normalization, as the V3/4 value was lower than 0.15. When total samples were considered, the pairwise variationV7/8 value was the lowest (0.165) but still above 0.15 (Figure 3).


Selection of appropriate reference genes for quantitative real-time PCR in Oxytropis ochrocephala Bunge using transcriptome datasets under abiotic stress treatments.

Zhuang H, Fu Y, He W, Wang L, Wei Y - Front Plant Sci (2015)

Pairwise variation (V) of 12 candidate reference genes calculated by geNorm to determine the optimal number of reference genes for accurate normalization. Asterisk indicates the optimal number of reference genes required for normalization.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4484982&req=5

Figure 3: Pairwise variation (V) of 12 candidate reference genes calculated by geNorm to determine the optimal number of reference genes for accurate normalization. Asterisk indicates the optimal number of reference genes required for normalization.
Mentions: The optimal number of the reference genes required for accurate normalization was determined by pairwise variation (Vn/Vn+1). In the subsets of drought and cold stress, the V2/3 value was below 0.15 (0.144 and 0.107, respectively), which suggested that two reference genes should be used for normalization. In the salt treatment subset, three reference genes were sufficient for accurate normalization, as the V3/4 value was lower than 0.15. When total samples were considered, the pairwise variationV7/8 value was the lowest (0.165) but still above 0.15 (Figure 3).

Bottom Line: To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable reference genes is required.When normalized with the most stable reference genes, the expression patterns of the three target genes were in accordance with those in the transcriptome data, indicating that the reference genes selected in this study are suitable.The usage of inappropriate reference gene would cause misinterpretation.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Resource Biology and Biotechnology in Western China, Department of Life Science, Northwest University Xi'an, China.

ABSTRACT

Background: Oxytropis ochrocephala Bunge, an indigenous locoweed species in China, poses great threats to livestock on grasslands. There is a need for further genetic study in the plants per se, for understanding the basis of its acclimation mechanism in various unfavorable environmental conditions and to implement effective control measures. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method for gene expression analysis. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable reference genes is required. The aim of this study was to select the most stable reference genes for transcriptional analysis in O. ochrocephala.

Results: We selected 12 candidate reference genes, 18S ribosomal RNA (18S RNA), actin2/7 (ACT7), β-actin (ACTB), actin101 (ACT101), actin11 (ACT11), β-tubulin (TUB), α-tubulin (TUA), glyceraldehyde-3-phosphate dehydrogenase-1 (GAPDH1), GAPDH2, metallothionein-like protein (MET), fructose-bisphosphate aldolase (FBA) and histone H3 (HIS), from the transcriptome datasets of O. ochrocephala and determined the suitability by analyzing their expression levels when exposed to a range of abiotic stress conditions. By employing software packages including geNorm, NormFinder and BestKeeper, HIS, ACT7, and ACT101 were assessed as the most suitable set for normalization in all samples. When normalized with the most stable reference genes, the expression patterns of the three target genes were in accordance with those in the transcriptome data, indicating that the reference genes selected in this study are suitable.

Conclusions: The study provided appropriate reference genes for accurate normalization in qRT-PCR analysis in O. ochrocephala and emphasized the importance of validating reference genes for gene expression analysis under specific experimental condition. The usage of inappropriate reference gene would cause misinterpretation.

No MeSH data available.


Related in: MedlinePlus