ed using our novel real-time RT-PCR assay. We assessed the sensitivity and PCR efficiency of our assay by titrating a broad range of cDNA template concentrations. EGFRvIII amplification 
was achieved with template amounts as low as 10 pg. The calibration curves demonstrate the robust linear dynamic range and high PCR efficiency of our novel assay; these are essential attributes for sensitive and precise real-time PCR assays. EGFRvIII Detection in Glioblastoma FFPE Tissue The frequency of EGFRvIII mutations in glioblastoma is well established in the literature. Consequently, we profiled EGFRvIII expression in tumors from a cohort of 26 glioblastoma patients to test the EGFRvIII detection performance of our realtime RT-PCR assay in FFPE tumor samples. For comparison, we also attempted EGFRvIII detection using MedChemExpress SB 1317 conventional RT-PCR and direct sequencing, in the same samples. Conventional endpoint RT-PCR was successful in only 7/26 samples, while target amplification failed in the remaining 19 cases. Direct sequencing gave slightly better results; 10/26 samples produced adequate quality electropherographic reads but electropherograms were inadequate for samples from the remaining 16 tumors. When performing our real-time RT-PCR assay on the glioblastoma samples, we used B2M as the reference gene. B2M was amplified successfully in all samples, while EGFRvIII was amplified in 10/26 samples. The detection of EGFRvIII by conventional RT-PCR and our novel real-time RT-PCR analysis were fully concordant; all samples that were EGFRvIII-positive by conventional RT-PCR were also EGFRvIII-positive according to our real-time RT-PCR analysis. Additionally, of the three tumor samples that were EGFRvIII-negative according to RT-PCR, two were re-classified as EGFRvIII-positive by our real-time RT-PCR assay; these tumor samples had high EGFRvIII Ct values and very high DCt values. The detection of EGFRvIII by our novel real-time RT-PCR method was fully concordant with the detection of EGFRvIII by direct sequencing in glioblastoma FFPE samples; all samples that tested positive for EGFRvIII by direct sequencing were also EGFRvIII-positive according to our novel real-time RT-PCR assay. Of the five samples that were EGFRvIII-negative by direct sequencing, two were re-classified as EGFRvIII-positive by our novel real-time RT-PCR method. These two samples only amplified a wild-type EGFR product by conventional RT-PCR but had high EGFRvIII Ct and DCt values when analyzed by our novel real-time RT-PCR. These results demonstrate the low February 2012 | Volume 7 | Issue 2 | e31723 EGFRvIII Expression in Oral Cancer sensitivity and inherent limitations of conventional PCR and direct sequencing methods when applied to FFPE tissue samples. Compromised RNA quality and fragmentation limits the ” size of amplicons that can be generated from FFPE tissue, thus rendering them unsuitable for conventional PCR and direct sequencing applications. Another possibility might be the low abundance of the EGFRvIII transcript in these samples that may be attributed to the tumor heterogeneity of glioblastomas. EGFRvIII Detection in OSCC FFPE Tissue We evaluated tumor samples from 54 OSCC patients for the presence of EGFRvIII using our novel real-time RT-PCR assay. February 2012 | Volume 7 | Issue 2 | e31723 EGFRvIII Expression in Oral Cancer 6 February 2012 | “ 25331948 Volume 7 | Issue 2 | e31723 EGFRvIII Expression in Oral Cancer control: U87MG and no template control: water. B. Sequencing of GBM 9 cD
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