Do or dye

A little bit of colour could be all that you need if you are struggling to aliquot liquids into multi-well plates when performing quantitative polymerase chain reaction assays

Most laboratory workers would agree that aliquoting transparent liquids manually into multi-well plates is one of the most challenging aspects of performing Quantitative Polymerase Chain Reaction (QPCR) assays - especially when the plates being used are manufactured from white plastic.

When opaque white plastic plates were introduced recently to the Thermo Scientific ABgene product range, users immediately saw an improvement in the sensitivity of their QPCR results - but they found it very difficult to visualise the master mix liquid when filling the wells by hand.

"When you are working with a plate of 96 or even 384 wells, it’s very easy to make errors. All it takes is for someone to interrupt you while you are in the middle of filling a plate"

This came as no surprise to the manufacturers, who were working with the white plastic plates in their own research labs. 

“Use of white plastic helps to return the fluorescent signal back to the QPCR detectors more efficiently than clear plates do, but this opaque background makes it very difficult to assess the accuracy of pipetting”, said Ian Kavanagh, research and development manager at Thermo Fisher Scientific. “When you are working with a plate of 96 or even 384 wells, it’s very easy to make errors. All it takes is for someone to interrupt you while you are in the middle of filling a plate with colourless liquid, and it’s almost impossible to go back and see exactly how far you have got. You can lift a clear plate up to try and see which wells you have filled, but this is obviously not an option with opaque white ones.  In fact, the only way to check if you have actually added liquid to a particular well is to put the pipette back in and try to get some liquid out again!”

In order to overcome this problem, Kavanagh and his team took up the challenge of developing a coloured master mix that would enhance the contrast between reagent and plastic without affecting the quality of the QPCR reaction in any way.

The result is ABsolute Blue QPCR master mix - whose clearly visible blue colour enables the status of master mix dispensing to be verified at a glance whilst enhancing performance with white PCR plastics.

The launch of ABsolute Blue is part of the ABgene product range of white plastic consumables which have been designed for use in QPCR applications, including gene expression and single nucleotide polymorphism (SNP) detection. This white plastic been shown to provide lower Ct values and lower variance than plates made from frosted or clear material, and it can eliminates the problem of cross-talk between wells1.  The visibility of ABsolute Blue against the white background has been achieved using an inert blue dye - one of more than 20 that were tested in Kavanagh’s laboratory.

“We needed to find a dye that could be visualised easily in white plastic or clear plates, but which would also not be inhibitory to the PCR process nor to the associated fluorescent emissions”, explained Kavanagh.  “We tried a lot of different colours, many of which were discarded because we found they inhibited the reaction.  In some cases, this inhibition disappeared when the dye was diluted down, but then of course the colour also became diluted.  So the initial challenge was to identify a suitable dye whose colour would be useful for visualisation.”

When filling plates with colourless liquid, spillage can occur

After short-listing a number of dyes, the next task was to establish whether their use would affect fluorescent emissions.  By checking to see whether the dyes they screened would interfere with the five channels and wavelengths normally used in QPCR, the team were able to narrow the choice down to a single candidate.

In the end, a single blue dye was found to be successful in fulfilling these key criteria.  This dye was included in the formulation of ABsolute Blue, along with various proprietary additives which help to deliver enhanced and consistent endpoint readings in addition to low Ct values. The ABsolute Blue also contains Thermo-Start DNA Polymerase - a chemically modified patented version of Thermoprime Plus DNA Polymerase that remains inactive until ‘switched on’ by high temperature incubation (hot-start), eliminating non-specific priming so that only the target DNA is amplified.  dTTP is also utilised in the dNTP mix for increased reaction efficiency. 

The development process for the mix took less than a year, after which it was successfully trialled at Oxford Brookes University School of Life Sciences by Simon Baker, a research fellow in biotechnology.  Baker and his colleagues were already very familiar with the difficulties inherent in visualising the manual aliquoting of transparent mixes, through working with them on a daily basis.  They knew that coloured dyes could be used in ordinary PCR master mixes, so they were excited by the prospect of seeing whether similar results could be achieved with QPCR.

“A blue master mix sample was supplied to us and we used this to fill 48 reactions - i.e. half a plate”, explained Baker.  “We then compared it with a colourless control.  Apart from the colour, we could not see any difference between the control and the blue dye mix at all, and there was no discernable difference in performance.  As a result, we are now using it routinely in our laboratory in preference to a clear mix.  It certainly helps us to avoid mistakes when we are loading plates by hand.”

When used in conjunction with white plastic plates, ABsolute Blue will help to ensure the consistency of results while minimising the risk of pipetting errors. It is normally recommended that the QPCR process should be repeated on a plate three times, and if one of those three replicates is very different from the others, all the data has to be discarded and the reaction performed over again. This is obviously very wasteful, and is something that should be ruled out by reducing pipetting errors. 

“As a company, we are always looking to identify simple but significant ways of reducing variance in a QPCR reaction, as this leads to enhanced sensitivity and efficiency”, added Melanie Yanek reagents product manager at Thermo Fisher Scientific. “By constantly seeking to minimise the variance in our consumables - whether reagents, plastics or seals - our aim is to improve the quality of our customers’ reactions.  In fact, we pride ourselves on responding rapidly to technological advances in the field of nucleic acid amplification.”

Reference
1. http://www.abgene.com/QPCR

By Melanie Yanek. Melanie is PCR Product Manager at Thermo Scientific, responsible for new product development of PCR plastics and reagents.