This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Carlos Noceda
University of Armed Forces-ESPE
Corresponding Author
ORCiD: https://orcid.org/0000-0001-6633-2698
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Backgroung
Selection of reference genes (RGs) for normalization of PCR-gene expression data includes two crucial steps: determination of the among-sample transcriptionally more stable genes and subsequent choosing of the most suitable genes as internal controls. Both steps can be carried-out through generally accepted strategies each having different strengths and weaknesses. The present study proposes to reinforce normalization of gene expression data by integrating and adding analytical revision at critical steps of those accepted procedures. Especially crucial is to counterbalance a higher representative number of RGs with a correspondent increase in their average transcriptional instability or a generalised co-expression trend among the samples. This methodological study used in vitro olive adventitious rooting as an experimental system, since the underlying morphogenetic process -wich is common to diverse species- is still not completely understood.
Results
Firstly, RG candidates were ranked according to transcriptional stability following a simple statistical method that reduces biasing effects of concomitant, systematic biological variations associated to experimental conditions, such as the variations caused by gene co-regulation. Those types of systematic co-variation are unconsidered by several popular ad hoc informatics programmes. To select the adequate genes among those already ranked, an algorithm of one of the ad hoc informatics programmes (GeNorm) was adapted to allow partial automatization of RG selection for any strategy of transcriptional-gene stability ordering. In order to delve into the resulting possible RG sets suitability for inter-assay comparisons and technical-error compensation, separate statistics were formulated. The achieved results were compared with those obtained by standard stability ranking methods. Finally, a double evaluation was performed to accurately contrast two choice RG sets. The whole strategy was applied to a panel considering several independent factors, but the suitability of the obtained putative RG sets was tested for cases restricted to fewer variables. H2B, OUB and ACT are valid for normalization in transcriptional studies on olive microshoot rooting when comparing treatments, time points and assays.
Conclusions
The set of genes identified as internal reference is now available for wider expression studies on any target gene in similar biological systems. The overall methodology aims to constitute a guide for general application.
Keywords
RT-qPCR, reference genes, expression stability, co-regulation, olive, adventitious rooting, alternative oxidase
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Carlos Noceda
University of Armed Forces-ESPE
Corresponding Author
ORCiD: https://orcid.org/0000-0001-6633-2698
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 19 Aug, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Backgroung
Selection of reference genes (RGs) for normalization of PCR-gene expression data includes two crucial steps: determination of the among-sample transcriptionally more stable genes and subsequent choosing of the most suitable genes as internal controls. Both steps can be carried-out through generally accepted strategies each having different strengths and weaknesses. The present study proposes to reinforce normalization of gene expression data by integrating and adding analytical revision at critical steps of those accepted procedures. Especially crucial is to counterbalance a higher representative number of RGs with a correspondent increase in their average transcriptional instability or a generalised co-expression trend among the samples. This methodological study used in vitro olive adventitious rooting as an experimental system, since the underlying morphogenetic process -wich is common to diverse species- is still not completely understood.
Results
Firstly, RG candidates were ranked according to transcriptional stability following a simple statistical method that reduces biasing effects of concomitant, systematic biological variations associated to experimental conditions, such as the variations caused by gene co-regulation. Those types of systematic co-variation are unconsidered by several popular ad hoc informatics programmes. To select the adequate genes among those already ranked, an algorithm of one of the ad hoc informatics programmes (GeNorm) was adapted to allow partial automatization of RG selection for any strategy of transcriptional-gene stability ordering. In order to delve into the resulting possible RG sets suitability for inter-assay comparisons and technical-error compensation, separate statistics were formulated. The achieved results were compared with those obtained by standard stability ranking methods. Finally, a double evaluation was performed to accurately contrast two choice RG sets. The whole strategy was applied to a panel considering several independent factors, but the suitability of the obtained putative RG sets was tested for cases restricted to fewer variables. H2B, OUB and ACT are valid for normalization in transcriptional studies on olive microshoot rooting when comparing treatments, time points and assays.
Conclusions
The set of genes identified as internal reference is now available for wider expression studies on any target gene in similar biological systems. The overall methodology aims to constitute a guide for general application.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
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