Wednesday, January 20, 2010

How Low Can You Go?

In a previous blog we described the use of The Epoch™ Multi-Volume Spectrophotometer System as a flexible instrument that provides micro-volume spectrophotometric quantification (2 µL sample volume with a nominal path length of 0.5 mm), microplate assays such as ELISAs and 1 cm pathlength spectrophotometric measurements. More recently, the analytical performance of micro-volume analysis, including limit of detection (LOD), was determined for nucleic acid samples. A summary of the results is provided below.

Limit of detection can be defined as the analyte concentration that provides a signal that is equivalent to three-fold the noise (standard deviation, sigma) in the background signal. This 3X-sigma signal is an optical density measurement that can be converted into an analyte concentration by determining the optical density of an analyte of known concentration, typically close to the detection limit. For DNA and RNA limit of detection determinations, water was used as the blank and the 3X-sigma signal was determined to be 0.0011 OD. A dsDNA standard of 4.7 ng/µL and an RNA standard of 4.3 ng/µL was used for the analyte limit of detection determination. Each of these standard concentrations was determined on the Epoch Multi-Volume Spectrophotometer System using the Take3 plate and BioCell – a 1 cm path length device. The average background corrected absorbance signal was 0.0042 OD for dsDNA and 0.0053 OD for RNA producing detection limits of 1.2 and 0.90 ng/µL, respectively.

Be on the lookout for similar analytical performance data for protein samples in the near future!

Are these nucleic acid detection limits sufficient for your laboratory needs or do you rely on the use of fluorescent reagents to quantify lower concentrations?

By, BioTek Instruments

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