Data are expressed as the mean +/- of [DELTA]Ct [+ or -] SEM relative to 16 h time point and normalised against the housekeeping gene
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
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.
Gene expression was standardized using the housekeeping gene
HPRT-1, and data was analyzed using the delta-delta-Ct method as previously described .
Apoptotic effects by different glucose level were determined by real-time, reverse transcription-PCR and GAPDH was use as the endogenous housekeeping gene
Target gene amplifications were normalized using housekeeping gene
expression levels of ATP synthase O subunit (ATP5O).
To ensure the integrity of these results, the additional housekeeping gene
18S rRNA was used as an internal standard to demonstrate that pbgd and actb mRNAs were not differentially regulated in the groups of GCs.