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Advisor(s)
Abstract(s)
In order to determine the suitability of reference
or housekeeping genes as internal controls in
real-time reverse transcriptase PCR (RT-PCR) assays
for quantification of target mRNAs, we studied the
levels of expression of four candidate reference genes
in maritime pine by real-time RT-PCR. The expression
levels obtained for glyceraldehyde-3-phosphate-dehydrogenase,
18S ribosomal RNA, eukaryotic translation
initiation factor eIF4AII and ubiquitin in nine stages
of embryo development revealed that none of the
genes tested proved to be suitable as an internal control.
Copy number quantification of the four transcripts
showed an average relative variation of seven
fold. We propose that the combination of a precise
method for RNA quantification, internal controls for
monitoring RT reaction and PCR efficiency and a
robust external standard curve can guarantee a reliable
absolute quantification of mRNA transcripts in real
time RT-PCR. This approach may avoid the controversy
in the use of housekeeping genes and may assume
special significance in tissues undergoing
developmental changes.
Description
Keywords
Housekeeping genes Maritime pine Plant embryogenesis Real-time PCR
Citation
Planta, 222, 556-563