Expression of reference gene(s) is expected to remain unchanged in fluoride-treated cells or in rodents relative to the corresponding untreated controls.
NormFinder is another approach to assess the stability of expression of reference genes [ 14].
Abundances of the 23 unigenes were calculated relative to the expression of reference genes DlFSD1a, EF-1a, and eIF-4a.
Both problem and calibrator samples were normalized by the relative expression of reference small non-coding RNAs (SNORD44 and SNORD48).
Results were normalized against the expression of reference genes ODC and RPL8 using CFX Manager software (BioRad).
They use different algorithms to analyse the variation in the expression of reference genes, and that may result in different recommendations for the most suitable reference gene.
This is an important characteristic of tests which utilize expression of reference genes to normalize expression of the test genes [ 12].
Equivalent expression of reference genes between tumour and normal tissues was then confirmed using a fold change cut-off of 3 [ 23].
In the pig however, very little is known about the expression of reference genes, certainly in combination with real-time RT-PCR.
Because of the inherent variation in the expression of reference genes the use of multiple reference genes rather than one reference gene is recommended to ensure reliable normalization of real-time PCR [ 10].
