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DNA Quantification: Comparison of UV Spectrophotometry and Picogreen Analysis

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DNA Quantification: Comparison of UV Spectrophotometry and Picogreen Analysis DNA Quantification from LGC DNA Quantification: Comparison of UV Spectrophotometry and PicoGreen Analysis Why do we need to quantify DNA? Following a DNA extraction procedure, To run KASP genotyping chemistry in it is good practice to quantify the our service laboratory, LGC requires concentration of the DNA sample prior 7.5 ng of good quality DNA per sample to using it in downstream applications. per SNP (based on human genome The input DNA requirements vary size); for detailed information on KASP between different applications, and DNA requirements for service projects accurate quantification is important to including adjustments for genome size, ensure that the end result is optimal. please view our factsheet. Quantification methods available, varying from traditional instruments that quantify DNA in plates or cuvettes, to There are a range of methods available for the instruments such as the NanoDrop (Thermo quantification of DNA samples. LGC utilise the following two methods: Scientific) that are designed to quantify DNA from micro-volumes of sample. • UV spectrophotometry (UV spec) – measures absorbance of the sample (at a wavelength • PicoGreen® – a fluorescent nucleic acid stain of 260 nm). A greater absorbance value that binds to double-stranded DNA (dsDNA). relates to greater quantities of nucleic acids. A higher level fluorescent signal indicates a A wide range of UV spectrophotometers are greater concentration of DNA. Quantification Advantages Disadvantages Quantification at LGC method Minimum Minimum volume detectable requirement concentration UV Spec Estimate of DNA purity is Potential overestimation of Plate reader Plate reader (Traditional also obtained (A260 / A280) dsDNA concentration: single- 50 µL 5 µg / mL plate reader stranded DNA (ssDNA) and and NanoDrop) RNA also absorb at 260 nm NanoDrop NanoDrop and can interfere with results 1 µL 2 µg / mL PicoGreen® Selectively binds to Method cannot be used to 10 µL of diluted 2 pg / mL dsDNA: ssDNA and RNA provide an estimation of DNA sample (typically do not interfere with results purity 1 in 1000 dilution) Laboratory testing and key findings 140 A set of 44 DNA samples, extracted in LGC’s 130 laboratory from human blood samples, were quantified using UV spectrophotometry (both 120 FLUOstar Omega plate reader and NanoDrop) 110 and PicoGreen methods, and the concentrations 100 concentration (ng / µL) obtained were compared. See Figure 1 for a A 90 comparison of the mean DNA concentrations 80 Mean DN obtained for all three methods. 70 UV Spec: NanoDrop UV Spec: FLUOstar PicoGreen Omega • Reported DNA concentration was significantly higher when quantified by UV spec (based on Figure 1. Mean DNA concentration of the 44 DNA samples 44 DNA samples, quantified in triplicate for as determined by both UV spectrophotometry methods and each method) PicoGreen® analysis • The mean UV spec value (NanoDrop) was For full details of the study, please view our 35% higher than that obtained by PicoGreen® Technical Note (available at www.lgcgroup.com/ • The mean UV spec value (plate reader) was genomics). 17% higher than that obtained by PicoGreen® • Repeatability of each method is comparable (based on average standard deviation values across samples, UV spec NanoDrop SD = 9 ng / µL, UV spec plate reader SD = 9 ng / µL, PicoGreen® SD = 11 ng / µL) No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording or any retrieval system, without the written permission of the copyright holder. © LGC Limited, 2014. All rights reserved. G2/CS/1214.
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