2001, 2008; Polanská et al. 2007). These studies also show that an alteration of the endogenous cytokinin content has a tremendous effect on morphological level (root/shoot formation, chloroplast ultrastructure) but also functionally (effect on photosynthesis, sink-source relationship). When analysing the expression stability of the nuclear- and plastid-encoded reference genes together, we saw that 18S rRNA (nuclear-encoded) and 16S rRNA (plastid-encoded) had the lowest stability in the geNorm analysis. These results clearly show that the use of ribosomal genes as internal standards is not advisable.
Nevertheless, 16S rRNA and 18S rRNA are frequently used as internal control in Northern blots (e.g. Covshoff et al. 2008; Soitama et al. 2008; Demarsy et al. TPCA-1 nmr 2006). Other click here drawbacks of the use of
ribosomal RNA as internal control are the high expression of levels of rRNA and the fact that ribosomal RNA expression is less affected by partial RNA degradation than other mRNA expression levels (Vandesompele et al. 2002). We also suggested RBCS as nuclear-encoded reference gene and saw that this gene had a very stable expression level. This gene is not commonly used as control gene as its expression levels were reported to vary greatly under different conditions (Sathish et al. 2007). Nevertheless, under our experimental conditions, this gene is very stable. Since chloroplasts have their own gene expression and a fraction of the proteins necessary for photosynthesis and protein synthesis are encoded within the chloroplasts while the remainder are encoded
in the nucleus, attention has to be paid when analyzing the gene expression of nuclear- or plastid-encoded genes. Normally, nuclear-encoded genes are normalized with nuclear-encoded reference genes and plastid-encoded genes with plastid-encoded Tau-protein kinase reference genes. However, it would be very interesting if normalisation of all these genes of interest was possible with the same reference genes. So, we selleck chemical investigated the effect of normalising some photosynthetic genes with nuclear normalisation factor (using Nt-SSU, Nt-ACT9, Nt-αTUB) or with plastid normalisation factor (using Nt-RPS3, NtNDHI and Nt-IN1). A difference in relative gene expression when using the two different normalisation factors was observed. We found that the gene expression of plastid-encoded PSBE, PSAA, PSAB and PETD diminished (except for PETD in 35S:CKX1) significantly when using the plastid normalisation factor compared to the calculated expression, using the nuclear normalisation factor. Also for the nuclear-encoded genes (ATPC and PSBO) there was an effect according to the used normalisation; however, the effect was not as pronounced as with the plastid-encoded genes of interest. This suggests that there is an effect of cytokinins on the expression level of the plastid-encoded reference genes.