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PCR & qPCRqPCR InstrumentationWhy are some Taq suited for fast cycling and others require the standard cycling profile?There are several commercially available Taq polymerases that differ in their enzymatic properties. Some display high processivity at temperatures above 60oC and are labelled as ‘fast cycling compatible’ polymerases because they can achieve PCR with a relatively short single annealing/extension step (<20 sec) without the need for a dedicated extension step at 68-72oC. These fast cycling compatible enzymes should be used with the ‘fast taq’ profile in the Q-qPCR software for details on how to set up a thermal profile. For Taq polymerases that are not compatible with fast cycling conditions, some consideration must be taken to select the appropriate thermal profile. For assays with an annealing temperature <60oC, a two-step cycling may not be achievable and a dedicated extension step and 68-72oC may be required. For assays with an annealing temperature >60oC, it may be possible to achieve a two-step cycling if the ‘standard taq’ profile is selected. This is because the standard taq setting will decrease the speed of transition between annealing and melt temperatures, which will increase the time that the reaction is held in the range of taq activity (a fast transition from annealing to melt temperature will quickly pass through this temperature range, while slowing this rate of increase will provide more time for taq to achieve amplicon extension).What is the ramp rate?We do not typically like to discuss ramp rates, except to point out that they are an arbitrary measurement that may be misleading. A ramp rate is the rate of temperature change over time, but there is no standardisation for how to take this measurement. For instance, you could measure a ramp rate for a metal component of a machine that in reality has no relationship with the heat over a block, and will certainly not reflect your sample reactions temperature. The lack of similarity in ways to define and/or measure ramp rates makes the calculated values not comparable (it is like comparing how much you like something. One person likes it heaps, another lots. Its meaningless to debate which person likes it more/less). The ramp rate for the Q is on our website, however it is much more indicative of machine performance to compare the time taken to complete a qPCR runHow fast can the Q complete a run?While this will vary according to your particular assay (different annealing temperatures and hold times), the Q can typically perform a 35 cycle qPCR run complete with melt curve in under 25 min.Why is the efficiency presented as a percentage and not a number between 1→2?qPCR efficiency reflects the similarity of data to exponential growth, or a doubling of signal for each qPCR cycle. The efficiency can be expressed as the rate of change of the data (as a multiplication factor), with 1 representing no change in data per cycle and 2 representing a doubling of the data per cycle. Alternatively, this can be expressed as a percentage of amplification efficiency, with 0% representing no change in the data per cycle and 100% representing a doubling of data per cycle.What are the benefits of using the REST analysis rather than ∆∆Ct?The REST analysis has the key advantage that it incorporates reaction efficiency while calculating expression ratios (Pfaffl 2002). The inability for the 2-∆∆Ct method to incorporate reaction efficiency could lead to major biases in the calculated expression ratios (eg see Ruijter 2009). While the 2-∆∆Ct method has been modified in some cases to account for amplification efficiencies derived from serial dilution of a standard sample, the combination of linREGPCR and REST allows the reaction efficiency of each actual sample to be used, eliminating the bias of the standard curve method that is often referred to as being only assay specific, and not sample specific (the standard curve method assumes that the calculated efficiency is both a perfect derivation of that value, and that the value perfectly matches that of the actual samples in each instance. The linREGPCR method includes sample specific differences in efficiency and does not assume equivalence of a standard curve with observed values, increasing the accuracy of the calculated data). Finally, the REST analysis uses non-parametric randomisation to calculate the error, which is more suitable for qPCR data than more standard parametric tests (eg T-test). The REST randomisation retains the same power as parametric testing without being affected by data distribution, unlike parametric tests which rely on normal distribution of data which is often not true of qPCR data, and also requires equal variance among the datasets which non-parametric testing does not.Why did you choose linREGPCR for data analysis?LinREGPCR uses sophisticated algorithms to increase the accuracy and reproducibility of qPCR data analysis, and is in our opinion the all-round best performing qPCR cycling analysis method. For more information, see (Ramakers 2003, ruijter 2009). The algorithm places the threshold value within the best portion of collected data (the window of linearity), and can scan for the threshold value with the least variation among and between samples to increase the accuracy of the data analysis. Importantly, linREGPCR will calculate efficiency values for each qPCR curve in the data analysis (meaning you don’t need to assume equal efficiency between standards and unknowns, and any sample specific efficiency differences are accounted for). linREGPCR employs a superior baseline correction method that has been shown to reduce the error in efficiency values that can be caused by using other baseline correction methods. Finally, the linREGPCR cycling analysis functions synergistically with our relative quantification analysis to provide the most accurate and reproducible gene expression ratio calculation.If my computer has an issue during a run – how can I retrieve my data?If a computer loses communication with the Q, the data from that run is stored on the machine. If you are using the same computer to reconnect to the Q-qPCR, clicking on the serial number icon (top right hand corner of the software) will give an option to ‘recover the run’ file. Similarly, if you connect to the Q-qPCR with a separate computer, you can still recover the data by clicking on the serial number icon, but now the option provided will be to ‘reconstruct the run’.Which fluorophores can I use with the Q?The Q-qPCR’s optical detectors will work optimally with FAM (green), Cal Fluor Orange 560 (yellow detector), Cal Fluor Red 610 (orange channel) and Quasar 670 (red channel). This combination of fluorophores will produce less than 3% crosstalk between any/all channel/s used. Other fluorophores with similar spectral properties will also be suitable. For example, Texas Red/ROX have very similar spectral properties to Cal Fluor Red 610, and can be measure on the orange channel. A list of popular fluorophores that are suitable for a particular detector are available while setting up the assay chemistry, and a more comprehensive chart is provided in the Q-qPCR manual (page 143).Can I export the raw data?Yes. To export the raw data, expand the save as icon and select the save as excel workbook option. This will save all of the information in the run file, including the raw data.Do I really need water balance tubes?Yes, for optimal performance the Q-qPCR should have 48x reactions of equal volume (samples or H20). This is because the amount of material that you need to heat/cool (ie the thermal load) will affect the amount of energy required to heat it to a particular temperature. As an example, a kettle filled with 1000 mL water will take more energy to heat to boiling than that same kettle filled with 100 mL. The Q-qPCR will run to specification when using a fully stacked rotor, but changes to the thermal load (incompletely filling the rotor) may impact the thermal performance.Can the Q-qPCR perform HRM analysis?Yes. The Q-qPCR has excellent thermal accuracy (uniformity of ±0.05oC) and can run a melt curve at 0.025oC/sec.Can the samples be used for post-PCR analysis when the tubes have the oil overlay?Yes, you can break through the oil layer with a pipette tip to remove your PCR reaction without the oil.What level of ROX should I use?The Q-qPCR does not require ROX. Other qPCR machines may require ROX to track temperature and/or optical performance due to thermal drift and variation in the detector setup. The use of magnetic induction (which has no thermal drift and will continue to provide uniform thermal performance over time), combined with the lack of moving parts in the detectors means that the Q-qPCR machine is highly reproducible over time. This means that while other machines may require the ROX channel just to monitor machine performance, the Q-qPCR has the advantage of allowing all channels to be available for real samples.Can I use the Q-qPCR for fieldwork?The Q-qPCR is well suited for fieldwork. You can run the machine from any power source, using a power inverter as required. Because you don’t need to calibrate for optics or temperature, and because of its small size/weight, the Q is ideal to transport to field sites while retaining functionality – it stays plug-and-play when other machines would need calibration.Can I configure the Q-qPCR software to easily enter sample names from a 96-well plate?Yes, the left hand side of the 96-well plate view will list the samples in the A1::H6 format, and the icon beside that will list the samples in the A7::H12 format to match the right hand side of a plate (the A1::H6 and A7::H12 icons are located at the top right hand corner of the samples editor). Next to these icons are the ’samples by column’ and ‘samples by row’ icons. If the samples were originally oriented according to the columns of a 96-well plate then you can simply enter these in to the Q-PCR software as it is the default display. If the samples were originally oriented across the rows of the 96-well plate, the samples will be located in different positions in the rotor, but once you select the ‘samples by row’ icon the samples selector will re-orient the display so that you can easily enter the sample namesCan I save my analysis settings to be automatically applied in subsequent runs?Yes. You can either click the ‘save analysis settings’ icon which is located at the top right hand corner of the analysis settings display (looks like an Erlenmeyer flask), or expand the assay in the navigation bar (left hand strip of the software) and click the + icon next to the analysis settings icon, fill out your information in the appropriate analysis setting and save (click on the assay name in the navigation pane and click the save icon).How can I find help about the Q-qPCR?The best initial resource is the Q-qPCR manual. You can access the manual by clicking on the ‘?’ icon located at the top left hand side of the software, and selecting the Q-qPCR manual option. Once open, you can scroll to the relevant section or use the ctrl+F search function to find what you need.