Selection of housekeeping genes
for gene expression studies in larvae from flatfish using real-time PCR.
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.
Bacterial identification is routinely carried out by 16S rRNA gene sequence analysis, supplemented with housekeeping gene
sequence analysis and phenotypic characteristics such as colony morphology, microbial physiology and biochemical analysis (14).
Tamas, "Housekeeping gene
selection in poplar plants under Cd-stress: comparative study for real-time PCR normalisation," Functional Plant Biology, vol.
In insects, the variation of housekeeping genes
has been already realized, and the effect of experimental conditions on the stability of housekeeping genes
was evaluated to select a most stable one as a reference (Bansal et al.
To avoid variations because of different sample amounts, standardization with housekeeping genes
was the method of choice.
Codon positions included were 1st + 2nd + 3rd + Noncoding for flaB sequences and housekeeping gene
Two housekeeping genes
, ACTB and PBGD (data not shown), were used to normalize sample-to-sample RT-qPCR assays.
Assay includes positive and negative controls as well as three sets of housekeeping genes
for normalization purposes.
The quality and quantity of the isolated RNA was confirmed with 2 housekeeping genes
, ACTB  (actin, beta) and HTN3 (histatin 3).
Usually, the well-known housekeeping genes
are chosen such as the glyceraldehydes-3-phosphate dehydrogenase (GAPDH), [alpha]-actins (ACTB), hypoxantine phosphoribosyltransferase (HRPT), [beta]-tubulin (TUB), elongation factor 1 alpha (EF1A) and 18S, 28S rRNA (Shu et al., 2004; Fernandes et al., 2008; Overgard et al., 2010).
In this work, to assess the existing genomic variability of 151 methicillin-resistant Staphylococcus aureus (MRSA) strains recovered from patients attended in two tertiary care hospitals, at the Medical School Hospital of Campinas State University (UNICAMP) in Campinas City and at the Medical School Hospital of Sao Paulo University in Ribeirao Preto City (FCMRP-USP), both cities located 155.34 miles apart in the Southeast region of Brazil, was used a combination of PCR-based techniques [(PCR amplification of spa and coa genes and housekeeping genes
(arcC, aroE, gmk, pta, tpi, yqiL)] with further restriction and fragment typing of the coa gene and housekeeping genes
(RFLP), plus the direct determination of the heterogeneity of the spa gene by agarose gel electrophoresis migration [3,6,19].