Only samples having a cycle threshold applying these ALB intron primers better than 35 were made use of for subsequent examination. Mutation screening PIK3CA mutations, PIK3R1 and AKT1 have been detected by sequencing of cDNA fragments obtained by RT PCR amplification. Exons to be screened during the 3 genes had been selected following mutational frequency described at COSMIC, Catalogue Of Somatic Mutations In Cancer. Screening by high resolution melting curve ana lysis was performed on PIK3CA exons one and 2, AKT1 exon 4 and PIK3R1 exons 11 to 15 on the LightCycler 480 applying LCGreen Plus Melting Dye fluorescence. Details of your primers and PCR ailments can be found on request. The amplified products have been sequenced using the BigDye Terminator kit on an ABI Prism 3130 automatic DNA se quencer with detection sensitivity of 5% mutated cells, and also the se quences had been compared with all the corresponding cDNA reference sequences .
All detected mutations have been confirmed during the second independent run of sample testing. Actual time quantitative RT PCR selleckchem RT PCR was utilized to the chosen genes and to TBP as endogenous mRNA handle. Primers are listed in Additional file 2, Table S2. PCR problems can be found on request. The RT PCR protocol employing the SYBR Green Master Mix kit on the ABI Prism 7900 Sequence Detection System is described in detail else where. The relative mRNA expression level of every gene, expressed since the N fold variation in target gene ex pression relative for the TBP gene, and termed Ntarget, was calculated as Ntarget 2Ctsample.
The worth with the cycle threshold of a given sample was determined by subtracting the common Ct value of the target gene from your typical Ct value of your TBP find out this here gene. The Ntarget values in the samples were subsequently normalized so that the median Ntarget value of typical breast samples was 1. Cut offs for normalized values 0. five and two. 0 were utilised to find out gene underexpression and overexpression, respectively. Immunohistochemistry PTEN and p85 protein expression levels have been assessed by immunohistochemistry staining on tumor sections from formalin fixed paraffin embedded blocks. Indirect immunoperoxidase staining was performed working with mouse monoclonal antibody directed towards human PTEN pro tein and rabbit polyclonal antibody directed against human p85 protein. The localization and in tensity of staining were assessed by two independent pa thologists blinded to real time RT PCR final results. Both antibodies have been used at a one 50 dilution. The im munohistochemical method was carried out as de scribed under, applying a water bath antigen retrieval method in every single situation. Sections have been mounted on pre coated slides and allowed to dry at 50 C overnight. Sections have been then dewaxed in xylene and hydrated by graded dilutions of ethanol.