The PCR reactions were carried out in 96 well plates that contained cDNA sample for target and housekeeping gene
according to the AB1 standard protocol.
As seen in this investigation, this approach can be inconclusive, and phylogenetic analysis of sequences including housekeeping genes
might be required.
For these strains, sequencing and comparing housekeeping genes
between maculicola pathovars, will point the genetic conservation between them or could establish the identity of another pathovar.
18S ribosomal RNA (18S rRNA) and elongation factor-1 alpha (EF1) were used as the internal control genes.
[DELTA][DELTA]Ct = (Ct of the sample of the target gene - Ct of the housekeeping gene
) - [DELTA]Ct (2)
Each tested strain has been analyzed by multilocus sequence typing based on sequences of seven housekeeping genes
, namely, gapA, infB, mdh, pgi, phoE, rpoB, and tonB.
Pre designed primers and probes for c-Myc gene were selected and purchased from Applied Bio System and for data normalization, GAPDH housekeeping gene
primer was custom designed using primer express software (ABI) (Table II).
Only 7 samples had counts for the housekeeping genes
below those considered acceptable and 3 samples positive for fusion-specific ALK probes but negative for the ALK 3'/5' imbalance were considered "inconclusive" (Fig.
TABLE 1: Housekeeping gene
PCR efficiency and stability.
Hypoxanthine guanine phosphoribosyltransferase (HPRT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as housekeeping gene
and data were normalized relative to the expression level of the HPRT gene.
Results were normalised relative to a housekeeping gene