Performance Data
Lyo-Ready Genotyping Direct qPCR FFPE combines the latest advances in buffer chemistry and PCR enhancers, together with an optimized antibody-mediated hot-start polymerase, dNTPs and MgCl 2 , along with a two-tube tissue extraction buffer, to release the DNA, for fast, precise, and highly reproducible allelic discrimination and cluster separation with SNP detection assays, even in the presence of PCR inhibitors presence in crude FFPE tissue extracts.
Higher DNA Extraction Yield with Meridian’s MDX168 Extraction Buffer
500 450 400 350 300 250 200 150 100 50 0
7 mg of FFPE lung tissue digested using MDX168 Extraction Buffer, Buffer ATL (Qiagen), Buffer G2 (Qiagen) and GeneJet FFPE Kit Extraction Buffer (ThermoFisher) and compared to purified DNA obtained using the Qiagen QIAamp FFPE Tissue DNA extraction kit (as a reference). The results show that a significantly higher quantity of DNA was obtained from tissue digested with MDX168 Extraction Buffer compared to purified DNA obtained with an extraction/ purification kit or tissues digested with other commercially available buffers. These results highlight the superior DNA extraction yield obtained from FFPE tissue with MDX168 Extraction Buffer. The DNA stored in the MDX168 Extraction Buffer is stable up to 24 hours at room temperature and 3 days at 4 °C.
MDX168 buffer
Buffer ATL Buffer G2
GeneJet Buffer
Purified DNA
Tighter fluorescence clusters with clearer allele discrimination compared to other commercially available mixes with FFPE samples KIT24 and PDGFRA (Platelet Derived Growth Factor Receptor Alpha) are both part of a receptor tyrosine kinases (RTKs) signalling pathway. Ataxia-telangietasia mutated (ATM) is a kinase that acts upstream of p53 and controls a DDR pathway critical to resolving double-stranded DNA breaks. Cancer-associated SNP mutations can be found in lung tumour tissue for all three of these genes. In the graphs below, A/ Lyo-Ready Genotyping Direct qPCR FFPE (MDX168), B/ KAPA Probe Force qPCR Mix (Roche) and C/ TaqPath TM ProAmp Multiplex Master Mix (ThermoFisher). The results illustrate the ability of Lyo-Ready Genotyping Direct qPCR FFPE to form tighter, more distinct clustering and therefore more accurate allelic discrimination in the presence of crudely extracted FFPE lung tissue than other genotyping kits.
KIT24_48 gene mutations
A)
B)
C)
T/T
C/T
C/C
RFU for Allele 1 - FAM
RFU for Allele 1 - FAM
RFU for Allele 1 - FAM
Healthy FFPE lung tissue (WT) and lung tumour tissue containing the cancer associated KIT24_48 mutation (Mutant) were digested using MDX168 extraction buffers. 5.2 ng of DNA from digested tissues were in the reaction. Homozygous Allele C ( red ) and allele T ( blue ) and heterozygous Allele C/T ( green ) with a NTC ( black ) and x for undetermined.
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