The specificity of any Real-Time PCR assay, whether TaqMan probe or SYBR Green I, is determined by the quality of the assay design. Non-specific amplification can occur for both SYBR Green I or TaqMan probe methods if the assay design is poor.
TaqMan assays will not generate a signal for any non-specific amplification whereas SYBR Green I assays might. However, non-specific amplification will affect the amplification efficiency and sensitivity of TaqMan assays in the same way as SYBR Green I assays, even though the amplification is not detected.
When designing primers for either system, it is important to avoid primer sets that generate any non-specific amplification products. With SYBR Green I assays, the ability to perform melt curve analysis is advantageous for primer design, as any non-specific amplification can be detected and identified in the melt curve. For TaqMan assays, detecting non-specific amplification usually requires another post-PCR analysis method such as agarose gel electrophoresis of the PCR products. Alternatively, the primers could be used in PCR with SYBR Green I and melt curve analysis performed after amplification to determine if any non-specific amplification occurs. This optimizes the primer design without the expense of a labeled probe.
The xxplates used in the system are custom designed for the xxpress® NGx. Both the xxplates and heat films are only available through BJS Biotechnologies approved distributors, check the website to find your local supplier.
The plates are in 24 well, 54 well or 96 well format with the same pitch and well size as standard Real-Time PCR plates. This means that you can use multichannel pipettes. Remember there are no hardware changes required to switch between these flexible formats.
The heat films are specifically chosen as their optical properties are compatible with the xxpress® NGx optics system.
Ramp rate is adjustable up to 10°C per second, the fastest available in the world.
You can export your data as .csv or RDML files.
The xxplates are sealed by welding on a heat film prior to performing the PCR. It is therefore not possible to re-use the xxplates. This would also enable contamination to take place.
The acquisition is done by a CCD camera through the top of the plate and through the optical heat seal.
RNA quality is perhaps the most important factor in generating reliable and reproducible Real-Time PCR data. Traditionally, RNA quality was assessed using gel electrophoresis and comparing the 28S and 18S ribosomal RNA bands. Gel electrophoresis is a laborious, time consuming, and low-throughput method that requires fairly large amounts of RNA.
Automated lab-on-chip capillary electrophoresis systems such as the Bioanalyzer (Agilent) and Experion (BioRad) have become popular tools for determining RNA quality. These systems use microfluidic technology to perform electrophoresis on glass chips at a miniaturized scale that overcome some of the issues of traditional electrophoresis. Data are presented as an electrophoretic trace of the RNA sample. The Agilent Bioanalyzer provides a quantitative measure of RNA integrity known as the RNA Integrity Number (RIN). A proprietary software algorithm examines the entire electrophoretic trace to determine RNA degradation and gives a numerical value between 1 and 10 to indicate RNA quality. An RNA sample with a RIN value of 10 is considered a highly intact sample where as a sample with a RIN value of 1 is considered a highly degraded sample.
The xxpress® centrifuge has a special rotor adapted to accommodate the xxplates.
In Real-Time RT-qPCR, genomic DNA can potentially be co-amplified during the PCR reaction, contaminating the sample and leading to erroneous results. To determine if an RNA sample is contaminated with genomic DNA it is important to include a no-reverse transcriptase control during the RT step, and all RT-qPCR experiments should include a no-RT control. If the RNA sample is free of genomic DNA contamination the no-RT controls should not generate any signal after Real-Time PCR.
The simplest and most commonly used method is the dilution or standard curve method. This method calculates PCR efficiency using the linear regression slope of a dilution series based on the following equation:
E = 10(-1/slope) -1
The ideal slope is -3.32, which correlates to an amplification efficiency of 100%, meaning exactly one copy per cycle. Slopes in the range of -3.60 to -3.10 are generally considered acceptable for Real-Time PCR. These slope values correlate to amplification efficiencies between 90% (1.9) and 110% (2.1). The correlation coefficient or R2 value should be at least 0.985 or higher.
The most widely used method to quantify RNA is traditional UV spectroscopy. A diluted RNA sample is quantified by measuring its absorbance at 260 nm and 280 nm. The concentration is calculated using the equation:
[RNA] ?g/ml = A260 x dilution factor x 40
where 40 is the average extinction coefficient for RNA
In addition, the A260/A280 ratio can be used to estimate RNA purity. An A260/A280 ratio between 1.8 and 2.1 indicates a highly pure RNA sample.
UV spectroscopy is relatively simple to perform but has several drawbacks. It does not discriminate between RNA and DNA so it is advisable to DNAse treat RNA samples before quantifying. DNA in the sample will lead to an overestimation of RNA concentration. Since proteins and residual phenol from the purification can interfere with absorbance readings, it is important to remove these contaminants in purification. Also, absorbance readings are dependent on pH and ionic strength. Dilute RNA samples in TE (pH 8.0) and use TE to blank the spectrophotometer before taking absorbance readings.
An alternative method for quantifying RNA samples is to use fluorescent dyes such as RiboGreen (Invitrogen). RiboGreen exhibits a strong fluorescent signal when bound to nucleic acids. Samples are quantified in a fluorescence microplate reader or standard spectrophotometer relative to a nucleic acid standard curve of known concentration. The linear range of quantification using RiboGreen is three orders of magnitude, from 1 ?g/ml down to 1 ng/ml. The major advantage of fluorescent dyes over absorbence-based methods is that it is not affected by contaminating proteins or organic solvents carried over from the purification process. DNAse treatment is still recommended as RiboGreen does not discriminate between RNA and DNA.
The LEDs are designed to outlast the life of the instrument.
You are unlikely to exceed the storage capacity of the xxpress® NGx. xxpress® NGx run files are small (~200–500 kb for a 40-cycle run),
In some cases it is possible to convert existing traditional PCR assays into Real-Time PCR assays, with a few considerations around primer design and master mix. Primer design is one of the first considerations for converting a traditional PCR assay. Real-Time PCR is most efficient with relatively short amplicon lengths, in the range of 50 to 150 bp. Longer products can be used if the cycling conditions are changed to accommodate longer extension times, but you should avoid products longer than 300 bp. In some cases it might be possible to design a TaqMan probe to hybridize between the two existing PCR primers. If not, you can use SYBR Green I for detection.
The master mix is another consideration when converting a traditional PCR assay into a Real-Time PCR assay. If a TaqMan probe can be designed, you might be able to use the same master mix that was used for the traditional PCR assay. If a TaqMan probe cannot be designed, you should add SYBR Green I to the master mix. In either case, a certain amount of optimization may be needed to obtain good Real-Time PCR results.
The xxpress® NGx optical system components do not move during operation, with the exception of the filter wheel, which moves while measuring the five emission wavelengths for each sample at each cycle.
The optical system is calibrated prior to shipment and we recommend that a recalibration is performed after 2500 runs or 1 year whichever occurs sooner. Pricing for this calibration plan is available from your local representative.
Yes, the xxpress® NGx system is compatible with the following dyes or their generic equivalents: FAM/SYBR, TAMRA, Texas Red, Cy5, Cy5.5.
|Channel||Excitation (nm)||Emission (nm)||Example Fluorophores Detected|
|1||474||507–527||SYBR Green I, FAM|
|2||535||567-583||JOE, HEX, VIC, TA|
|3||578||617-633||Texas Red, ROX|
Some basic Real-Time PCR terms and their definitions are:
Amplification plot – Plot of fluorescent signal versus cycle number.
Baseline – The initial cycles of PCR where there is little to no change in fluorescence.
Threshold – The arbitrary level of fluorescence used for Cq determination. Should be set above the baseline and within the exponential growth phase of the amplification plot.
Cq (quantification cycle) – The fractional cycle number where fluorescence increases above the threshold. Also referred to as Ct (threshold cycle) or Cp (quantification cycle).
R – Reporter signal.
Rn – Normalized reporter signal.
?Rn – Baseline subtracted normalized reporter signal.
Slope – Indicates the efficiency of the reaction. With 10-fold dilutions, a slope of -3.32 indicates a perfect doubling of product per cycle (100% PCR efficiency).
R2 – Reports the linearity of the standard curve.
One advantage of Real-Time PCR over traditional PCR is that it is a closed-tube system requiring no post-PCR processing. Real-Time PCR has higher precision, increased sensitivity (down to one copy), increased dynamic range (greater than 8 logs), and high resolution (less than two-fold differences).
Multiplex Real-Time PCR can be applied to relative quantification experiments where the gene of interest and reference gene are co-amplified in the same reaction. Multiplex Real-Time PCR can also be used for allelic discrimination assays, where two differentially labeled probes detect two alleles of a single nucleotide polymorphism. Another application of multiplex Real-Time PCR is pathogen detection, where multiple pathogens can be detected in one reaction.
Real-Time PCR has been used in quantification of gene expression, viral quantification, validation of array data, pathogen detection, and allelic discrimination.
The starting materials for Real-Time PCR can be RNA, genomic DNA, or plasmid DNA. RNA must be reverse-transcribed into complementary DNA (cDNA) before PCR. More recently, kits have become available that can perform RT-qPCR (RT = reverse transcription) directly from cells without the need for a separate RNA synthesis step.
There are two major detection chemistries used for Real-Time PCR: enzymatic and hydrolysis (TaqMan) probe-based chemistries, and DNA-binding SYBR Green I dye-based chemistry. Additional detection chemistries include Molecular Beacons, Scorpions probes and LUX primers.
There are three phases of PCR amplification: exponential, linear, and plateau. The exponential phase is the first phase of PCR amplification. Reaction components are in excess, there is an exact doubling of product each cycle, and the reaction is specific and precise. Real-Time PCR measures the Cq value at this phase of PCR. The linear phase is the second phase of PCR amplification. The reaction components are being consumed, amplification slows, and the reactions become highly variable. The final phase of PCR amplification is the plateau phase. The reaction is complete and no more products are being generated. Traditional PCR takes its measurements during this phase of PCR.
xxpress® NGx is an open platform that supports all current Real-Time PCR detection chemistries including DNA binding dyes, hydrolysis probes, hybridisation probes, as well as other detection chemistries such as molecular beacons and Scorpion primers.
xxpress® NGx does not use PMTs or photodiodes, it uses a CCD camera as the detector.
Real-Time PCR uses various fluorescent detection chemistries that allow you to monitor the PCR reaction as it progresses. The amount of fluorescent signal generated is directly proportional to the amount of DNA being synthesized during the PCR reaction. Data are collected at each cycle as opposed to traditional PCR, which collects data at the end of the reaction. This allows samples to be characterized by when amplification is first detected as opposed to the amount of product generated after PCR cycling. The greater the amount of the target sequence, the earlier amplification will be detected.
It is a powerful post-PCR analysis technique used to identify the DNA products present. During PCR reaction dyes are incorporated into the DNA which fluoresce in the presence of double stranded DNA. During a melt the DNA is subjected to a rise in temperature which causes it to denature. As more of the DNA becomes single stranded the fluorescence decreases. A particular DNA product will have an associated temperature at which it denatures and therefore loses its fluorescence.
Multiplex Real-Time PCR is a technique in which multiple target sequences are amplified and detected in a single PCR reaction. Amplified sequences are distinguished by the use of different dyes conjugated to the probes. The number of targets that can be detected in a single reaction is technically limited only by the availability of spectrally distinct dyes and the ability of the Real-Time PCR instrument to effectively excite and detect those dyes. Some advantages of multiplex Real-Time PCR are reduced reagent costs, reduction in sample use, and increased throughput.
Developing multiplex Real-Time PCR assays can be difficult and time-consuming. As the reaction complexity increases, significant optimization may be required to generate reliable data. It can be a challenge to develop multiplex assays that amplify all targets with equal efficiency.
When developing multiplex Real-Time PCR assays, you need to consider primer design, the relative expression levels of target sequences, and master mix / reagent conditions.
Use the same design criteria for each primer/probe set and screen all sequences against each other to determine any potential primer-dimer formation. In addition, perform a BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine primer specificity.
If the expression levels of the target sequences are significantly different, the most abundant target will be preferentially amplified and deplete all the reaction components, compromising amplification of the less abundant targets. One way to address this issue is to limit the primer concentrations of the most abundant target, using the lowest primer concentration that produces the same Cq and PCR efficiency. Limiting the primer allows the most abundant target to amplify and go to completion without depleting all the reagents needed for the other sequences.
Amplifying multiple target sequences creates additional demand for reaction components. Taq DNA polymerase, Mg++ and dNTP concentrations may need to be optimized to improve amplification of all targets. Master mixes optimized specifically for multiplex Real-Time PCR are now commercially available, and can reduce the amount of time required for optimization.
Compare experiment data that uses a single assay with the experiment data when the assay is combined into a multiplex assay. Sensitivity and PCR efficiency needs to be the same in both types of assay use.
The temperature resolution of the xxpress® NGx system is 0.1° C.
The xxpress® NGx optical system supports five channels:
|Channel Number||LED Colour||Peak Excitation (?)||Emission Wavelength band(?)|