Quant-iT™ PicoGreen® dsDNA Assay Kit From Molecular Probes (Invitrogen)

The Molecular Probes PicoGreen® assay is a fast, simple and reliable method to quantify double-stranded DNA in samples. Although the assay is capable of quantifying DNA from any source (cells or tissues), my experiences are with quantifying DNA extracted from osteogenic cultures which are known to produce a thick, mineralized matrix.

Assay preparation is minimal and the key reagents are included in the kit. Cell lysis in water using a freeze-thaw process is adequate to release DNA before proceeding with the assay. The reaction itself consists of mixing 25 µl of the sample with 25 µl of TE buffer and 50 µl of the working solution of PicoGreen® dye in a 96-well plate. The reaction is then incubated in the dark for 5 minutes to permit the dye to bind to double stranded DNA. The fluorescence of the samples is then measured on a plate reader at excitation/emission of 435/535 nm. Although the dye has an extremely large linear range, it may be necessary to dilute your sample. Pre-testing will allow you to determine the optimal volume of lysis reagent with which to process your cells or tissue.

A key benefit of the assay is that the dye has a very low binding efficiency for non-double stranded nucleic acid (especially RNA). As a result, there is minimal interference from non-genomic nucleic acid. While most other molecular components of the cell lysate do not interfere with dye binding, larger particulate cell debris or matrix components may block light paths when the plate is measured on your plate reader and consequently reduce accuracy of your results. If your cultures are particularly confluent or produce a lot of matrix, it may be necessary to pellet cell debris before processing, or use alternative lysis reagents which are compatible with the dye. In short term assays (such as dose responses), pelleting debris is not as essential and omitting this step simplifies the protocol in high-throughput screening situations.

Although the dye reagent is available separately, the kit version of the product comes with the buffers required (except for lysis buffer) and DNA standards to allow for absolute quantification of DNA.

I have not compared the quantification of DNA between PicoGreen® and other spectrometric methods. However, if you have pure samples of DNA, it will probably be more cost effective to simply spec your samples in a reliable spectrometer. I would only recommend the PicoGreen® dye when your sample has not been further processed to remove cellular proteins and other contaminants.