Selection of reference genes for qPCR- and ddPCR-based analyses of gene expression in Senescing Barley leaves.
Leaf senescence is a tightly regulated developmental or stress-induced process. It is accompanied by dramatic changes in cell metabolism and structure, eventually leading to the disintegration of chloroplasts, the breakdown of leaf proteins, internucleosomal fragmentation of nuclear DNA and ultimately cell death. In light of the global and intense reorganization of the senescing leaf transcriptome, measuring time-course gene expression patterns in this model is challenging due to the evident problems associated with selecting stable reference genes. We have used oligonucleotide microarray data to identify 181 genes with stable expression in the course of dark-induced senescence of barley leaf. From those genes, we selected 5 candidates and confirmed their invariant expression by both reverse transcription quantitative PCR and droplet digital PCR (ddPCR). We used the selected reference genes to normalize the level of the expression of the following senescence-responsive genes in ddPCR assays: SAG12, ICL, AGXT, CS and RbcS. We were thereby able to achieve a substantial reduction in the data variability. Although the use of reference genes is not considered mandatory in ddPCR assays, our results show that it is advisable in special cases, specifically those that involve the following conditions: i) a low number of repeats, ii) the detection of low-fold changes in gene expression or iii) series data comparisons (such as time-course experiments) in which large sample variation greatly affects the overall gene expression profile and biological interpretation of the data.
Molecular and Systems Biology Department, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland; Institute of Computing Science, Faculty of Computing, Poznan University of Technology, Poznan, Poland.
Agnieszka
A
Zmienko
Institute of Computing Science, Faculty of Computing, Poznan University of Technology, Poznan, Poland.
Anna
A
Samelak-Czajka
Molecular and Systems Biology Department, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
Michal
M
Goralski
Department of Plant Physiology, Faculty of Biology, Adam Mickiewicz University, Poznan, Poland.
Ewa
E
Sobieszczuk-Nowicka
Molecular and Systems Biology Department, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
Piotr
P
Kozlowski
Molecular and Systems Biology Department, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland; Institute of Computing Science, Faculty of Computing, Poznan University of Technology, Poznan, Poland.
Marek
M
Figlerowicz
D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D001467:Hordeum / Q000235:genetics*; D018515:Plant Leaves / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D020871:RNA Stability; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors