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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 35-40 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.
We have not screened for any other components to add to the Lyo-Ready Probe 1-Step Mix apart from primers, probes and ROX. If you would like to add extra lyo-compatible components, you will need to test their activity in both the wet mix and the lyophilised material, as well as assess their stability over the time. PCR Biosystems is not responsible for any loss of performance caused by the added component.
Can ROX be added to the Lyo-Ready Probe 1-Step Mix and can it have a negative impact on the reaction?
Yes, ROX (6-carboxy-X-rhodamine), available separately, can be added to the kit and it would not interfere with the lyophilisation process.
ROX 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 template 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.
No, the 3200x UltraScript RTase provided in the kit already 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@example.com 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
The Lyo-Ready Probe 1-Step Kit contains 2 components: 4x Lyo-Ready Probe Mix and 3200x UltraScript Reverse Transcriptase (RTase). 1.25 μL of 3200x UltraScript RTase should be added for each mL of 4x Lyo-Ready Probe Mix used. Due to the high concentration of the RTase, the volume supplied is very small, so we recommend spinning the tube every time before pipetting the desired volume of enzyme.
Whilst the 2 components of the kit are stable and have a longer shelf life (see expiry date of each component), the 4x Lyo-Ready Probe 1-Step Mix (obtained by adding the 3200x UltraScript RTase to the 4x Lyo-Ready Probe Mix) should be used immediately or can be stored for up to 3 days at 4 °C. We therefore recommend preparing only the amount of 4x Lyo-Ready Probe 1-Step Mix required and to return the remaining separate components to storage at between -30 °C and -15 °C.
Is it normal if the fluorescence of 4x Lyo-Ready Probe Mix 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.
4x Lyo-Ready Probe Mix contains MgCl2 at a concentration of 18 mM. This means the final concentration in the reaction is 4.5 mM.
Gene specific primers should be used in the 1-step reaction.
In the product manual we are providing a general lyophilisation protocol, tested for 2 mL glass vials filled with 500 μL of the Lyo-Ready Probe 1-Step Mix. We observed that lyophilisation is facilitated (i.e., the program is shorter, and cakes have a more uniform aspect with no signs of shrinkage) when the Lyo-Ready Probe 1-Step Mix is diluted to 1x or 2x (with oligonucleotides and water), so we recommend doing this dilution before starting the run.
Depending on the volume lyophilised, the material of the vials (i.e., glass vs plastic) and the lyophiliser used, the cycle may be optimised.
If the cakes are not properly formed after the cycle, we recommend repeating with a more conservative cycle, in which the primary drying is carried at -50 °C and time is extended to guarantee the correct cake formation. Secondary drying phase could also be extended if required.
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. Normally the concentration of the probe is half of that of the amplifying primers, but optimisation and validation are required for each primer set.