QuantiFluor® dsDNA System

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Exposure to light will reduce the sensitivity of the assay. Store QuantiFluor® dsDNA Dye and working solution protected from light

Upstream tips
Exposure to light will reduce the sensitivity of the assay. Store QuantiFluor® dsDNA Dye and working solution protected from light

Publication protocol

FFPE sample selection
To assess pre-PCR variation, engineered, cell-line FFPE curls (Horizon Diagnostics, Cambridge, UK) were employed. SW48 and MCF10A cell lines were genetically modified to contain clinically relevant EGFR and BRAF mutations using recombinant adeno-associated virus vectors. The modified cell line was titrated against its matched normal parental cell line to generate mixtures containing quantifiable amounts of EGFR and BRAF mutations. Cells were grown under standard tissue culture procedures, trypsin was used to release the cells and cells were counted using a NC100 Nucleocounter (Chemometec, Denmark). The cells were used to generate FFPE blocks manufactured using a validated, proprietary method to achieve cell numbers of 160 000 cells per FFPE section. Therefore, each FFPE section contained defined theoretical DNA yields (see figure 1 for details). Cell numbers per section were validated theoretically using known core and cell size. Section volume (V=πr2h) was calculated as 15 µm * π * (2500 µm2)=294 375 000 µm3. SW48 and MCF10a cell volume (V=4/3πr3) was calculated as (4/3) * π * (7.5 µm3)=1766 µm3. The potential number of cells comprising each 15 µm section is therefore 166 690 cells. Consequently, the theoretical DNA yield of each 15 µm section is 1.1 µg assuming 6.6 pg DNA content in each cell (theoretical DNA content in diploid cells). This is in accordance with Horizon Diagnostics Aperio (Leica Biosystems, Denmark) cell count data that measured cell numbers across 24 FFPE blocks, demonstrating a mean cell count of 167 681 with an SD of 35 993. Additionally, comparison of DNA yields from 15 µm sections cut from 12 original FFPE blocks was performed by Horizon Diagnostics. The Maxwell DNA extraction kit (Promega, USA) was used to extract DNA from a total of 36 FFPE sections. DNA quantitation was performed using the Quantifluor assay (Promega, USA). The mean DNA yield per section was 465 ng with an SD of 94, which is in keeping with the calculated theoretical yield. The allele ratios of mutations in each sample were quantified using droplet digital PCR (ddPCR) performed with the BioRad QX100 platform (Hercules, California, USA).

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Manufacturer protocol

Download the product protocol from Promega for QuantiFluor® dsDNA System below.

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