Quantitative PCR (qPCR), either by real-time or end-point measurement, is a useful technique for comparing the expression level of a certain gene in different samples. It’s widely accepted in therapeutic research, pathogen quantification, cancer studies, etc. Similar to any other quantitative analysis, a piece of convincing qPCR data addressing a specific underlying theory should show a change with measurable order of magnitude, with statistical significance in at least 3 replicates, and should be reproducible. In order to obtain reliable data, many researchers may focus mainly on the selection of the qPCR detection system (machinery and dye), the PCR reagents, and the design of primers and probes. However, they may ignore the container of their precious reactions – the PCR plate, and what the signals must pass through to be detected – the plate sealer.
Bio-Rad iCycler iQ™ PCR plates, 96-well, 0.2 ml (Cat#2239441), with cut away corner at bottom left corner (near H1 well) is perforated every three columns for easy set-up of triplicate reactions. It is recommended to be used with Bio-Rad Microseal® ‘B’ film plate sealers (Cat#MSB1001); these are optically clear, adhesive seals, with perforation for end-tab removal. I would say the designer of the layout of these 2 products understands what those performing qPCR look for. When preparing as many as 96 reactions, the perforation every three columns really helps to reduce errors in adding the template to triplicates. The sealer with perforated end-tabs provides higher flexibility in that the tabs can be removed for researchers using semi-skirted plates. These details look minor but they suit the end-users’ need ideally.
However, despite the good design, I found several flaws in these products. The perforated end-tabs of the sealer are quite difficult to tear off. Moreover, although the manufacturer claims that the sealer is ‘strongly adhesive for reliable sealing’, the statement is true before heating only! I dare say the plate and the sealer are an ‘imperfect match’. The most annoying fact is that the two materials (made for the sealer and the plate) expand unequally upon heating, which results in their detachment from one another during the qPCR run.
This is a nightmare for qPCR: evaporation in random wells causes artifactual fluctuation in Ct values between replicates. Once you identify the wells with a higher chance of evaporation, you will avoid using them. Thus the ‘perforation every three columns’ design is completely useless. It’s disappointing that using three products from the same manufacturer (iCycler iQ™ qPCR system, the plate and the sealer, all from Bio-Rad) cannot give reliable results. And this plate is MORE expensive than other brand names in my region.
With this fatal flaw, there are a number of outliers in almost every run I do, and this happens to every labmate in my group. It’s a pity that the poor selection of the manufacturing materials wastes the good design.