Data are expressed as the mean +/- of [DELTA]Ct [+ or -] SEM relative to 16 h time point and normalised against the
housekeeping gene GAPDH.
MLST, as defined by Urwin and Maiden, is a genotyping tool based on the sequences of
housekeeping genes that evolve slowly.
(2004) Real-time RT-PCR analysis of
housekeeping genes in human skeletal muscle following acute exercise.
For this test, real-time PCR was used employing relative quantification, using [beta]-actin as
housekeeping gene, to normalize the data obtained.
New primers were designed to determine the expression levels of the detected 4 pil genes and the in-house designed bracket gene in comparison to the rpoD gene that was selected as a
housekeeping gene. Selection of rpoD is in concordance with Savli et.
The expression of the gene-of-interest was determined relative to the expression of the
housekeeping gene (62 microglobulin) which is not thought to change significantly with illness (12,13).
This could be due to an ineffective
housekeeping gene, GAPDH.
The data obtained by RT-qPCR are typically normalized with an internal control or
housekeeping gene. The use of an internal control allows the correction of some systematic errors that may occur during the process such as initial size of the sample and pipetting errors, quantity and quality of RNA obtained, changes in enzymatic efficiencies, or presence of inhibitors (Bustin 2002, Vandesompele et al.
Apoptotic effects by different glucose level were determined by real-time, reverse transcription-PCR and GAPDH was use as the endogenous
housekeeping gene (D, E).
Target gene amplifications were normalized using
housekeeping gene expression levels of ATP synthase O subunit (ATP5O).