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Why Not A Block?
Most real-time systems have adapted an optics subsystem onto a standard 96-well or 384-well thermal cycler. The result is unavoidable thermal variation across the block (Fig 1) and differences in illumination and optical signals detected from each sample (Fig 2). This well-to-well variability is the most challenging but important issue affecting real-time instrument performance and has been well-documented in 96-well systems. For example here and here.
Variability problems are only exacerbated by the new trend to speed-up cycling times. Increasing speed only worsens thermal performance because the faster a block is heated or cooled, the greater the well-to-well thermal variation observed. A good new (2008) publication on fast block cycler performance is located here. |
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In addition, most real-time systems use Peltier devices to actively heat and cool samples. The Rotor-Gene doesn’t use these devices, a distinct advantage because they are expensive to repair and, like light bulbs, can fail unpredictably. They can also produce localized “hotspots” as device junctions inevitably begin to fail.
Well-to-well optical variability is the main reason so many real-time systems require extensive set-up calibration and ongoing recalibration as light sources are replaced or begin to age. Furthermore, normalization of signal levels from every well during every experiment is also usually necessary. This is normally done using a housekeeping fluorophore (for example the ROX™ passive reference dye). A major benefit of the Rotor-Gene design is that optical calibration and passive reference normalization is simply not necessary. |
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Fig 1: Color-enhanced thermal contour map of a metal 96-well block. The block is heated and cooled using Peltier devices surface-mounted to the underside. The gradient is from red (hot) to blue (cool). Dissipation of heat from the Peltier devices is not uniform across the block (”edge effect”) or vertically from the base to the top of each sample well. In addition, localised hot-spots can also occur (as shown) when individual Peltier device junctions begin to fail.
The hotter region at the top edge of the sample wells originates from heat transferred from a typical “heated lid” mechanism used to help curb sample condensation effects. The Rotor-Gene uses centrifugal force to continually remove trace sample condensation. |
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Fig 2: Color-enhanced optical brightness contour map from a block based real-time amplification system. Shown is the camera signal collected from a clean but empty 96-well silver block. The color gradient is from red (bright) to blue (weak). The color scale reflects intensity changes in the monochromatic signal. This particular system uses a projector lamp for illumination and a CCD camera to record signals. Note the variation across the block despite correction through a complex Fresnel lens.
Images like this of an empty block are often used as part of routine block maintenance to detect “hot” wells having contaminant fluorphore (from a marker pen or spill, for example). Hot wells (not shown) can obscure an individual result. By contrast, the Rotor-Gene does not have wells to clean or maintain since reaction tubes hang in mid-air.
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Fig 3: Thermal hot and cold spots in a typical 96-well block system.
Shown is colour-enhanced signal collected using a DriftCon® RF (CyclerTest), a multi-well probe thermal verification system. Data was generated from simultaneous measurements taken at 15 locations across the block. The colour gradient is from red (hot) to blue (cold). Data shows how the hottest spot is not always in the middle of the block and that edge wells are not always the coldest locations, as often assumed. In addition, the hot and cold regions change when the instrument switches between heating and cooling modes. The DriftCon system captures dynamic data allowing thermal overshoot and undershoot of temperature non-uniformity to be determined. This data was collected five seconds after reaching 95°C. This is considered a "good" result by standards and is a genuine picture from a real test.
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