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Ct values can vary between template concentrations, reaction optimisation, instruments and laboratories so care must be taken when selecting a cut-off Ct value. Generally, Ct values over 35 would begin to be considered unreliable. However, late Cts could be observed for inefficient reactions using low template copy number. It is always good practice to normalise cut-offs with relative or absolute quantification methods. It is also recommended to run and analyse melt curves or gels of the products to determine products of any late amplifications
ROX (6-carboxy-X-rhodamine) is used as a passive reference dye in ROX-dependent real-time PCR instruments to normalize for variations of fluorescence levels that can arise mainly due to optical path variations among wells. Normalisation of the fluorescence intensity (Rn) is done in real-time PCR software by dividing the emission intensity of the specific signal by the emission intensity of ROX.
ROX does not take part in the PCR reaction and its fluorescence levels are not proportional to the quantity of DNA in each well, so the addition of this fluorophore to a mix provides a constant fluorescent signal during amplification.
Different types of real-time PCR instruments requiring a passive reference standard have different optimal concentrations of ROX, mainly due to the different optical configurations of each system (i.e. the different type of excitation source and optics used).
The addition of either too little or too much ROX would result in a very noisy signal impacting on the results of the reaction. Therefore, it is extremely important for the user to:
- Determine the correct ROX concentration to optimise real-time PCR results, and
- Check the ROX settings on the software used to set up the reaction
A useful selection tool for the most commonly used systems can be found here.
We recommend using a minimum of 2 minutes for activating the polymerase. Longer times of up to 15 minutes can also be used with no detrimental effects to the enzyme.
Both the cDNA synthesis and PCR reactions occur in the same mix. This option is suited for high throughput applications due to its speed and ease of set up. There is also a reduced risk of contamination. It is not ideal for low quality RNA samples or if the cDNA is required for archive or separate analysis.
The cDNA synthesis and PCR reactions occur separately. This option is better suited if the cDNA product needs to be retained for analysis. It also allows for higher levels of reaction optimisation. It permits control over the type and concentration of enzyme, RNA input and concentration of cDNA which, in turn, results in higher sensitivity compared to the 1-step format.
No, the 20x UltraScript RTase contains an RNase inhibitor to prevent any degradation and increase sensitivity.
If you are observing unusually late Ct values, try diluting the template RNA. By doing this, you are diluting any inhibitors which may be present to a concentration where they do not inhibit the reaction. Additionally, try increasing the reverse transcription step to 55 °C and increasing the annealing/extension temperatures. This may help resolve difficulties caused by secondary structures present in the RNA template and/or primers.
In cases where reaction inhibition may be involved, try reducing the amount of template1 or add 0.4 – 4.4 mg/ml BSA to the reaction2.
For more specific problems contact [email protected] with the following information:
- Amplicon size
- Reaction setup
- Cycling conditions
- Screen grabs of amplification traces and melting profile
1 Scipioni et al. A SYBR Green RT-PCR assay in single tube to detect human and bovine noroviruses and control for inhibition. Virology Journal.5:94 (2008). doi: 1186/1743-422X-5-94
2 Plante et al. The use of bovine serum albumin to improve the RT‐qPCR detection of foodborne viruses rinsed from vegetable surfaces. Applied Microbiology. 52:3 (2010) doi: https://doi.org/10.1111/j.1472-765X.2010.02989.x
Is it normal if the fluorescence of qPCRBIO Probe Mixes differs from the one obtained with competitors’ products?
Different products could give a different plateau of fluorescence. However, this has no impact on quantification accuracy and Ct values will not differ among products.
Probe based kits such as qPCRBIO Probe 1-Step Virus Detect offer higher sensitivity and are unlikely to show non-template amplifications. Multiplexes can be measured using amplicons with different fluorophores for specific probes, which cannot be achieved using dyes.
Dye-based systems such as qPCRBIO SyGreen 1-Step Detect and 1-Step Go detect any intact dsDNA and will therefore show primer dimers and off-target/non-template amplifications. These can be separated from product peaks by analysing melting curves.
ROX is a passive reference dye which means it does not take part in the PCR reaction. It is used to normalise non-PCR related fluctuations in fluorescence. You can use our qPCR Selection Tool in the Resources section to determine which of our qPCR mixes are best suited for your qPCR machine.
All 4x qPCRBIO Probe 1-Step Virus Detect mixes contain MgCl2 at a concentration of 18 mM. This means the final concentration in the reaction is 4.5 mM.
Gene specific primers can be used in the 1-step reaction.
What should be considered when doing multiplex amplification with qPCRBIO Probe 1-Step Virus Detect?
The primers should be designed to ensure that they have similar annealing temperatures, are specific to the target, and do not form primer dimers. The length of the reverse transcription phase can be extended to 10 minutes to ensure there is enough template for priming and amplification.
We recommend using 400-1000 nM of each primer. There is a degree of flexibility around this recommended concentration, however the primer concentration should not be increased beyond this range as this may significantly affect the activity of the enzyme.