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Validation of reference genes for quantitative gene expression studies in Volvox carteri using real-time RT-PCR

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Abstract

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is a sensitive technique for analysis of gene expression under a wide diversity of biological conditions. However, the identification of suitable reference genes is a critical factor for analysis of gene expression data. To determine potential reference genes for normalization of qRT-PCR data in the green alga Volvox carteri, the transcript levels of ten candidate reference genes were measured by qRT-PCR in three experimental sample pools containing different developmental stages, cell types and stress treatments. The expression stability of the candidate reference genes was then calculated using the algorithms geNorm, NormFinder and BestKeeper. The genes for 18S ribosomal RNA (18S) and eukaryotic translation elongation factor 1α2 (eef1) turned out to have the most stable expression levels among the samples both from different developmental stages and different stress treatments. The genes for the ribosomal protein L23 (rpl23) and the TATA-box binding protein (tbpA) showed equivalent transcript levels in the comparison of different cell types, and therefore, can be used as reference genes for cell-type specific gene expression analysis. Our results indicate that more than one reference gene is required for accurate normalization of qRT-PCRs in V. carteri. The reference genes in our study show a much better performance than the housekeeping genes used as a reference in previous studies.

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Acknowledgments

The authors thank Kordula Puls for technical assistance.

Conflict of interests

The authors declare that they have no competing interests.

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Authors and Affiliations

  1. Department of Cellular and Developmental Biology of Plants, University of Bielefeld, Universitätsstr. 25, 33615, Bielefeld, Germany

    Arash Kianianmomeni & Armin Hallmann

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  1. Arash Kianianmomeni
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  2. Armin Hallmann
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Correspondence to Arash Kianianmomeni.

Electronic supplementary material

Below is the link to the electronic supplementary material.

11033_2013_2784_MOESM1_ESM.docx

Supplemental Figure S1. RNA sampling and diversity of samples. Supplemental Figure S2. Separate analysis of each sample pool by geNorm – Average expression stability during stepwise exclusion of the least stable candidate reference gene. (DOCX 718 kb)

Supplemental Table S1. The ten candidate reference genes – Primer sequences and amplicon characteristics. (DOCX 18 kb)

11033_2013_2784_MOESM3_ESM.docx

Supplemental Table S2. Detailed list of Ct values of the ten candidate reference genes at all investigated biological or experimental conditions. (DOCX 31 kb)

Supplemental Table S3. Stability values of candidate reference genes as calculated by NormFinder. (DOCX 12 kb)

Supplemental Table S4. BestKeeper analysis of candidate reference genes using Ct values as input. (DOCX 15 kb)

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Kianianmomeni, A., Hallmann, A. Validation of reference genes for quantitative gene expression studies in Volvox carteri using real-time RT-PCR. Mol Biol Rep 40, 6691–6699 (2013). https://doi.org/10.1007/s11033-013-2784-z

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  • DOI: https://doi.org/10.1007/s11033-013-2784-z

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