Figure 1. A comparison of results from qPCR using clear and white plastics. (A) Compared to the clear wells, the white wells result in lower Ct values (higher sensitivity). (B) The white wells also exhibit increased consistency among technical replicates.
PCR/qPCR Plastics Considerations–4 Main Attributes
PCR is a sensitive and efficient method for amplifying a single copy of a target DNA sequence to millions of copies, in a short time. Therefore, PCR plastics used for the reaction must be not only free of contaminants and inhibitors but also of high quality to enable optimal PCR performance. As a user it is important to understand the features and terminology associated with PCR/qPCR plastics, as explained below, so that you can choose the right plastics to obtain accurate and reliable experimental data (Related: Plastics selection guides).
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1. Plastics composition
PCR/qPCR plastics are most commonly made of polypropylene, because it is inert and able to withstand rapid changes in temperatures during thermal cycling. The inert property of polypropylene minimizes absorption of the reaction components, helping to ensure optimal PCR results. To further warrant lot-to-lot consistency in purity and biocompatibility, high-quality virgin polypropylene, like that used for medical-grade or molecular biology–grade plasticware, should be used in manufacturing. Note that some PCR/qPCR plates are made with a stronger polycarbonate frame for compatibility with high-throughput robotic applications.
PCR tubes and plates are often available in a variety of colors to facilitate visual organization and identification of samples, especially for high-throughput experiments. Although the color of the plastic has no impact on DNA amplification, in setting up reactions for real-time PCR or qPCR, white and/or frosted plastics may be recommended over clear to enable sensitive and accurate fluorescence detection (Figure 1). Users should follow recommendations of the qPCR instrument manufacturer for color considerations.
White plastics improve the sensitivity and consistency of qPCR data by preventing fluorescence refraction out of the tube (Figure 2A). With refraction being minimized, more signal is reflected back to the detector, increasing the signal-to-noise ratio (Figure 2B). In addition, the white wall minimizes variation across technical replicates (Figure 1B) by preventing transmission of fluorescence signals to the thermal cycler block where it can be absorbed or inconsistently reflected. On rare occasions, where the fluorescence detector is oversaturated from increased signal reflection of the white well, frosted wells may be a better alternative.
Figure 2. (A) White wells prevent refraction of fluorescence signals and their absorption by the thermal block in qPCR experiments. (B) White plates reflect significantly more signal than clear plates. Three dilutions of fluorescein were added to the plates for comparison.
Note that ultraclear caps or optical seals should be used to seal qPCR tubes and plates in order to enhance sensitivity and reduce variability, since they are specifically designed to allow passage of fluorescence signals without interference.
2. Manufacturing overview
a. Preventing dust and biological contamination
To avoid contaminants that can interfere with DNA amplification, PCR/qPCR plastics should be free of nucleases and DNA. Although sterilization methods like autoclaving and irradiation destroy bacteria and DNases, these approaches alone cannot remove dust and DNA residue. The remaining dust particles may inhibit PCR, and fragmented DNA can still act as templates for nonspecific amplification.
Therefore, the manufacturing of the plastics, from molding to final packaging, should be carried out in facilities that limit particle counts in the environment. For example, a class 100,000 cleanroom allows no more than 100,000 particles of ≥0.5 μm in diameter in a cubic foot of air. These types of facilities adhere to stringent guidelines set forth by the International Organization for Standardization (e.g., ISO 9001) to prevent dust and biological contamination.
b. Quality assurance tests
For quality assurance, PCR/qPCR plastics may be visually and/or physically examined by the manufacturer for their integrity in protecting reaction components. Physical tests can be performed to assess evaporation, sealing, and electrical continuity of wells to check for leaks. For biological testing, the plastics may undergo qPCR amplification of certain DNA targets to assess contamination by nucleases and human DNA. The manufacturer may provide a Certificate of Analysis for PCR/qPCR plastics that have undergone such stringent tests.
3. PCR/qPCR plastics essentials
PCR/qPCR plastics come in a number of sizes and forms. Understanding their features and related terminology can help you achieve optimal PCR and qPCR results when using these plastics.
a. General attributes
i. Reaction size or fill volume
The fill volume of PCR/qPCR plastics determines the size (volume) of a PCR/qPCR reaction that can be successfully performed. Overfilling a vessel can result in inefficient heat transfer, spills, and cross-contamination. On the other hand, underfilling may lead to sample loss due to evaporation. The thermal block also dictates the size of plastics to be used and their optimal fit for integrity and efficient heat transfer. The most common volume sizes of PCR/qPCR vessels are:
- Tubes: 0.5 mL, 0.2 mL, 0.1 mL
- 96-well plates: 0.2 mL, 0.1 mL
- 384-well plates: 0.02 mL
The profile of a PCR/qPCR vessel refers to its height, so a “low profile” means the height is reduced (Figure 3). The shorter design of low-profile plastics minimizes the air space above the reaction, reducing the effects of evaporation and enhancing thermal conductivity. Therefore, the low-profile option is recommended when it is available. Low-profile plastics are generally required for fast thermal blocks and thus may sometimes be referred to as “fast” tubes or plates.
iii. Wall thickness
PCR/qPCR plastics with uniform, thin walls maximize consistent heat transfer to and from the reaction samples. Ultrathin-wall plastics, which are approximately 50% thinner than the standard version, further reduce the thermal barrier, leading to faster and more robust reactions.
The caps and seals of the plastics for qPCR should be of high optical clarity to maximize passage of fluorescence signals and minimize distortion. Note that white vessels are recommended for improved qPCR data, as explained above.
b. Tube formats
Tubes are an ideal choice for PCR/qPCR experiments of low to medium throughputs. Individual tubes and tube strips are two of the most common formats available (Figure 4).
- Individual tubes provide the flexibility of setting up the exact number of reactions to run. Also, the 0.5 mL size is available only in individual tubes when a scale-up reaction is needed.
- Tube strips are commonly available as strips of 8 or 12 with separate or attached caps. Designed to work with both single-channel and multi-channel pipettes, tube strips are suitable for low- and medium-throughput experiments. Those with attached caps offer the option of opening and closing the tubes independently to prevent sample contamination.
With certain thermal cyclers that exert strong lid pressure for efficient PCR, a special tray/retainer may be required to prevent crushing of the tubes and tube strips.
c. Plate formats
PCR/qPCR plates usually come in formats of 96 wells and 384 wells, and to a lesser extent, 24 wells and 48 wells (see also plastics segmentation). The thermal cycler being used and the nature of applications being performed will determine whether the following features of PCR/qPCR plates are suitable for your experiments (also see our plastics selection guide).
A “skirt” of a PCR/qPCR plate is a peripheral panel around the plate (Figure 5). A skirt provides stability to the plate during pipetting in the reaction setup, and mechanical strength for better handling by robotic instruments. PCR/qPCR plates can be categorized as nonskirted, semi-skirted, and fully skirted.
- A nonskirted plate lacks the surrounding panel (Figure 5A). This format fits blocks of most thermal cyclers and real-time PCR instruments, but it is not suitable for robotic applications.
- A semi-skirted plate has a short panel around the edge of the plate (Figure 5B), providing adequate support during pipetting and mechanical strength for robotic handling.
- A fully skirted PCR/qPCR plate has a full-height panel surrounding the plate (Figure 5C). This plate format is designed to fit securely over a thermal cycler with a raised block (which facilitates robotic handling). The full skirt also enhances mechanical strength, making it excellent for use with robotic platforms in an automated workflow.
Figure 5. Nonskirted, semi-skirted, and fully-skirted PCR/qPCR plates.
The deck of a plate refers to its top surface (Figure 6).
- The flat deck design provides a universal fit with most thermal cyclers and facilitates sealing and handling.
- The raised deck design is required for optimal fit and performance with some thermal cyclers and instruments, helping to balance the lid pressure without the need for adapters.
The notch is a modified corner of a PCR/qPCR plate, which is determined by the instrument being used for optimal fit. The notch may be at the H1, H12, or A12 position of a 96-well plate, or the A24 position of a 384-well plate (Figure 7).
Figure 7. PCR/qPCR plates with notch corners.
iv. ANSI/SBS footprint
To enable compatibility across different high-throughput systems with robotic liquid handling, PCR/qPCR plates should adhere to standard dimensions set forth by the American National Standards Institute (ANSI) and the Society for Biomolecular Sciences (SBS, now part of the Association for Laboratory Automation and Screening (SLAS)). Plates with the ANSI/SBS format maintain standard footprint, height, well positions, etc., thereby streamlining the automation process.
v. Well rim
A raised rim around the wells is a common feature of PCR/qPCR plates (Figure 8). This design enables secure sealing with film and safeguards against evaporation.
Figure 8. Raised well rims of a PCR/qPCR plate.
Alphanumeric lettering on PCR/qPCR plates helps identify the location of individual wells and keep track of samples. Raised letters are printed, often in white or black, for easy visualization (Figure 9A). In automation, however, raised letters may interfere with the sealing process. For automated applications, engraved lettering (Figure 9B) may improve sealing of the outer edges of the plate.
Figure 9. Alphanumeric lettering of PCR/qPCR plates. (A) Raised (printed) lettering. (B) Engraved lettering.
Some 96-well PCR/qPCR plates are designed with segmentation. This design allows for a full 96-well plate to be easily broken into smaller plate sizes when the whole plate is not used, reducing consumable waste and cost. Plates with 24-well segmentations are most common, but 32-well and 8-well segmentations are also available for convenience and flexibility (Figure 10).
Semi-skirted and fully skirted PCR/qPCR plates may be barcoded for easy recording and tracking in high-throughput experiments (Figure 11). For secure and reliable tracking, barcode labels should be scratch-resistant and be able to withstand chemical exposure and a wide temperature range (e.g., –196°C to 120°C). The position of a barcode reader may vary by the automated platform, and 4-way barcoding or custom options are alternative solutions in those cases.
Figure 11. Barcoding. (A) PCR/qPCR plate with a barcode on one side. (B) Common barcode formats.
The two most common types of PCR seals are cap strips and adhesive films. Cap strips may be used to seal both PCR/qPCR tube strips and plates, but films are designed to seal PCR/qPCR plates (Table 1). To obtain sensitive and reliable qPCR data, caps and films that are intended for use in qPCR should have high optical clarity with equal thickness and transparency.
Table 1. PCR/qPCR sealing caps and films.
Note that the caps and films must seal the tubes and plates securely to prevent sample evaporation during thermal cycling. Tools such as sealing applicators and capping tools are available to facilitate proper sealing.
Cap strips are the most common form of PCR/qPCR caps to seal tube strips and 96-well plates (Figure 12). In addition to fit, clarity and shape are two main features to consider in choosing the caps.
- Although clarity of the caps has no impact on DNA amplification, ultraclear or optical caps are important for collecting reliable data in qPCR experiments.
- The top of the cap may be flat or domed. The choice between the two depends on the lid design of the thermal cycler being used.
- If the lid has a slightly concave inner surface to prevent deformity of the tubes, domed caps help ensure fit for better PCR results while acting as a pressure absorber to maintain the integrity of the tubes.
- Flat caps provide a surface for labeling and are designed for thermal cyclers with flat lids. Optical caps for qPCR assays are often flat to allow undistorted passage of fluorescence signals.
Sealing films for PCR plates may be made of clear plastic or aluminum foil, each offering unique benefits (Figure 13).
- Adhesive clear films may have a nonsticky surface that seals the wells only after sufficient pressure has been applied on them, allowing resealing of the plates and leaving well openings clear for maximum fluorescence transmission when necessary. qPCR films must be of high optical clarity for minimal impact on data collection. For sensitive applications like qPCR for human identification, sealing films must be treated with ethylene oxide by the manufacturer (ISO 18385 compliant) to eliminate possible traces of DNA that may yield false-positive amplification.
- Although unsuitable for qPCR, adhesive aluminum films are ideal for light-sensitive samples and are pierceable for sample transfer in PCR.
4. Throughput of PCR/qPCR experiments
The experimental throughput of a PCR/qPCR assay may determine the type of plastic vessel to select for optimal handling. For low- to medium-throughput applications tubes are better suited in general, whereas for medium- to high-throughput experiments plates are more ideal. Some plates are designed for flexibility of throughput in mind, such as with segmentation into single-row tube strips (learn more: segmentation).
For high-throughput automation, the plate’s skirt and material composition are two of the most important aspects to consider, because of the requirement for the plate to be handled by the “grippers” or paddles of the automation systems (Figure 14A).
- Semi-skirted and fully skirted plates provide necessary side surfaces for the grippers to grasp, whereas nonskirted plates are not suitable for automation (learn more: plate skirts).
- Despite its durability, the polypropylene of standard PCR plastics is not rigid enough to withstand forces exerted by the grippers. Plate warping can impede automated processes and interfere with collection of downstream data. Therefore, PCR/qPCR plates for automation should be constructed with a stronger polycarbonate frame to resist distortion caused by the robot grippers and to better tolerate rapid heating and cooling. The wells should still be thin-walled polypropylene wells for efficient heat transfer to the reaction mixture (Figure 14B).
Note that frames of multiple colors can help with organization and visual monitoring of laboratory assays in a high-throughput setting.
Figure 14. (A) Robotic grippers of a liquid handling system. (B) Construction of a PCR/qPCR plate for high-throughput/robotic applications.
In conclusion, as a critical component in the setup of PCR, plastics play an important role in the success of the experiments and data collection, especially for mid- to high-throughput workflows. Understanding the basics of PCR/qPCR plastics when selecting them can help you achieve optimal PCR and qPCR results.
For Research Use Only. Not for use in diagnostic procedures.