Fig 1.
Design of multiplex qPCR for KRAS genotyping.
In a first step was performed PCR to amplify the Non-Mutated reference amplicon KRAS (NM) and simultaneously Multiplex Allele specific (Mult-AsP). When both amplicons were amplified (NM and Mult-AsP), the sample was interpreted as positive and seven allele specific primer (AsP) PCR reactions for the specific identification of mutation were performed. If no amplicon is amplified in the Mult-AsP reaction but amplified in the non-mutated reference reaction, the sample was interpreted as negative or wild type.
Fig 2.
AsP and Mult-AsP PCR assays for analysis of the mutational status of KRAS codons 12 and 13 in reference samples.
Genomic DNA of seven reference standards harboring KRAS mutations in codons 12 and 13, was used for AsP and Mult-AsP PCR assay. In all cases the qPCR assays contained the non-mutated reference control reaction (red line). Each reference DNA was amplified in AsP and Multi-AsP PCR (green and blue lines, respectively). In gray curves indicated NTC reaction.
Fig 3.
Allele-specific primer only amplify sequence-specific templates.
Genomic DNA harbouring G12V or G12V KRAS mutations were used for AsP and Mult-AsP PCR assays, whenever indicated.
Table 1.
ΔCq values of AsP and Mult-AsP qPCR assays.
Fig 4.
KRAS multiplex mutation analysis with colorectal carcinoma FFPE.
Genomic DNA from FFPE tissues were used for AsP and Mult-AsP PCR assay. In all cases the qPCR assays contained the non-mutated reference control reaction (red line). Each DNA was amplified in AsP and Multi-AsP PCR (green and blue lines, respectively). In gray curves indicated NTC reaction.
Table 2.
KRAS mutation analysis relative to NM reference DNA in colorectal carcinoma FFPE tissues.
Table 3.
Sensitivity of the KRAS Multiplex qPCR assay.