Tissue-specific evaluation of suitable reference genes for RT-qPCR in the pond snail, Lymnaea stagnalis
- Published
- Accepted
- Subject Areas
- Genetics, Molecular Biology, Neuroscience
- Keywords
- gene expression, normalization, mollusc, gastropod neurobiology
- Copyright
- © 2019 Young et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2019. Tissue-specific evaluation of suitable reference genes for RT-qPCR in the pond snail, Lymnaea stagnalis. PeerJ Preprints 7:e27695v1 https://doi.org/10.7287/peerj.preprints.27695v1
Abstract
Reverse transcription quantitative PCR (RT-qPCR) is a robust technique for the quantification and comparison of gene expression across multiple tissues. To obtain reliable results, one or more reference genes must be employed to normalize expression measurements among treatments or tissue samples. Candidate reference genes must be validated to ensure that they are stable prior to use in qPCR experiments. The pond snail (Lymnaea stagnalis) is a common research organism, particularly in the areas of learning and memory, and is an emerging target for qPCR experimentation. However, no systematic assessment of reference genes has been performed in this animal. Therefore, the aim of our research was to identify stable reference genes to normalize gene expression data from a variety of tissues in L. stagnalis. We evaluated a panel of seven reference genes across six different tissues in L. stagnalis with RT-qPCR. The genes included: elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (ACTB), beta-tubulin (TUBB), ubiquitin (UBI), prenylated rab acceptor protein 1 (Rapac1), and a voltage gated potassium channel (VGKC). These genes exhibited a wide range of expression levels among tissues. The stability of each of the genes was consistent when measured by any of the standard stability assessment algorithms: geNorm, NormFinder, BestKeeper and RefFinder. Our data indicate that GAPDH and EF1α are highly stable in the tissues that we examined (central nervous system, tentacles, lips, penis, foot, mantle) as well as in pooled analyses. We do not recommend VGKC for use in RT-qPCR experiments due to its relatively low expression stability. Our results were generally congruent with those obtained from similar studies in other molluscs. Given that a minimum of two reference genes are recommended for data normalization, we suggest GAPDH and EF1α are a strong option for multi-tissue analyses of RT-qPCR data in Lymnaea stagnalis.
Author Comment
This is a submission to PeerJ for review.
Supplemental Information
Expression levels for each gene in each tissue based on Cqs
Box plot: upper and lower box limits indicate 25th and 75th percentiles, dark/light grey interface indicates the median, ‘x’ indicates the mean, and whiskers indicate the minimum/maximum values. Data was collected from six biological replicates with three technical replicates for each.
GeNorm stability rankings for all candidate reference genes sorted by tissue
Genes are ranked in order from the least stable to the top two most stable.
NormFinder stability rankings for all candidate reference genes sorted by tissue
Genes are ranked in order from the least stable to the most stable.
BestKeeper stability rankings for all candidate reference genes sorted by tissue
Genes are ranked in order from the least stable to the most stable.
Representative image of PCR products for each reference gene
The agarose gel was 2% and contained a no primer control (NPC).