qPCRBIO Probe 1-Step Go

All qPCRBIO Probe 1-Step Go mixes contain MgCl₂ at a concentration of 9 mM. This means the final concentration in the reaction is 4.5 mM.

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:

1. Determine the correct ROX concentration to optimise real-time PCR results, and
2. 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.

The PCRBIO 1-step Go RT-PCR Kit has been developed for endpoint RT-PCR. The qPCRBIO Probe 1-Step Go and qPCRBIO SyGreen 1-Step Detect|Go are our probe- and dye-based options for real-time RT-PCR, respectively.

1-step

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.

2-step

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, this kit will not work for 2-step reactions. For 2-step reactions, we recommend the UltraScript® cDNA Synthesis Kit (PB30.11) or UltraScript® 2.0 cDNA Synthesis Kit (PB30.31) for more difficult targets, along with real time PCR kits depending on your needs.

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.

Gene specific primers can be used in the 1-step reaction.

Yes, qPCRBIO Probe 1-Step Go can be used for micro RNA templates. Although we do no sell dedicated kits, all of our RTases can be used for miRNA quantification and analysis.

We advise that you use one of the two following approaches:

• Use universal RT primers and add poly(A) or poly(U) tails (e.g. by poly(U)-polymerase), followed by cDNA synthesis using universal primers^1,2.
• Use specific RT primers and omit the tailing step^1,3-5.

If you are unfamiliar with the specifics of those approaches, please refer to the reference list below, which serve as a guideline.

1  Dave, V. P. et al. MicroRNA amplification and detection technologies: opportunities and challenges for point of care diagnostics. Lab Invest 99, 452-469, doi:10.1038/s41374-018-0143-3 (2019).

2  Mei, Q. et al. A facile and specific assay for quantifying microRNA by an optimized RT-qPCR approach. PLoS One 7, e46890, doi:10.1371/journal.pone.0046890 (2012).

3  Chen, C. et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33, e179, doi:10.1093/nar/gni178 (2005).

4  Raymond, C. K., Roberts, B. S., Garrett-Engele, P., Lim, L. P. & Johnson, J. M. Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs. RNA 11, 1737-1744, doi:10.1261/rna.2148705 (2005).

5  Androvic, P., Valihrach, L., Elling, J., Sjoback, R. & Kubista, M. Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification. Nucleic Acids Res 45, e144, doi:10.1093/nar/gkx588 (2017).

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 under the Resources drop-down menu to determine which of our qPCR mixes are best suited for your qPCR machine.

Probe based kits such as qPCRBIO Probe 1-Step Go 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 | 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.

No, the RTase Go contains an RNase inhibitor to prevent any degradation and increase sensitivity.

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

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 primer/probe concentrations should not require adjustment unless the expression levels are significantly different. For highly expressed targets, the amount of primers should be decreased, and for targets expressed at lower levels it should be increased.

We recommend using 0.4 µM of each primer. There is a degree of flexibility around this recommended concentration however, the primer concentration should not be increased, as this may significantly affect the activity of the enzyme.

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.

mRNA isolation is not normally required. qPCRBIO Probe 1-Step Go Kit has been developed to work on samples containing as little as 1 pg total RNA or 0.01 pg mRNA.

If you’re working with rare mRNA species, use a sequence-specific primer in the RT reaction to increase the 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