Sequence based prediction of 26S rRNA in medicinal plants for qRT-PCR purposes
Keywords:
housekeeping genes, sequential prediction, plant tissue stabilityAbstract
Reverse transcription quantitative real-time PCR is worlwide most often used for the analysis of relative expression levels of genes under various experimental treatments or in various comparative studies. Methodological approach of qRT-PCR offers advantages such as high reproducibility and sensitivity. To obtain the very precise differences in expressions of genes of interest in qRT-PCR, stably expressed reference genes are selected for standard correction. Different stably expressed reference genes have been reported for plants, but establishing the characterics for them stil continuous. Here, bioinformatic based prediction of conserved part of 26S rRNA was performed as the sequence is not available in public databases for one of the analysed species. Subsequently, its transferability was tested for three different medicinal species – Pulmonaria officinalis, Plantago lanceolata and Hedera helix. Sequence alignment resulted in a primer pair that was tested for its specifity in the real-time PCR analysis of all of species used in the study and for three plant parts – leaf, stem and flower. The amplicon of 26S rRNA was confirmed in all of analysed medicinal plant species and a its stability in tested tissues was proved. The most stable results were obtained for Hedera helix, but in all analysed species, the differences of expression of 26S rRNA was very low.
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