Lyo-Ready Probe Mix and Probe 1-Step Kit

Ct values can vary between template concentrations, reaction optimization, instrumentation, and laboratories so care should be taken when selecting a Ct cut-off value. Generally, Ct values above 35-40 will start to be considered unreliable. However, late Ct values may be observed for inefficient reactions with low template amounts. The best practice is to normalize cut-offs with relative or absolute quantification methods. It is also advisable to run and analyze melting curve or gel profiles of the products to determine product from any late amplification.

Yes, ROX (6-carboxy-X-rhodamine), supplied separately, can be added to the kit and it will not affect lyophilisation.

ROX is used as a passive reference dye in ROX-dependent real-time PCR instruments to normalize for fluctuations in fluorescence levels which may appear due largely to optical path change between wells. Normalization of fluorescence intensity (Rn) is performed in real-time PCR software by dividing the emission intensity of the specific signal with the emission intensity of ROX.

ROX does not participate in the PCR reaction and its fluorescence levels do not correlate with template amounts in each well; thus, adding this fluorophore to the mix provides a consistent fluorescent signal during amplification.

Different real-time PCR instruments require passive reference standards with differing optimal ROX concentrations, mostly due to each system's different optical configurations (i.e., different excitation and optics types used).

Adding too much or too little ROX will result in a very noisy signal adversely affecting the reaction results. Therefore, users should:

1. Identify the correct ROX concentration to optimize real-time PCR results, and
2. Check the ROX settings on the software used to set up the reaction

A useful guide for choosing the appropriate system is available here.

If you observe unusually late Ct values, try diluting the sample RNA. By doing so, you are diluting any inhibitors that may be present at concentrations that inhibit the reaction. Additionally, try increasing the reverse transcription step to 55 °C and increasing the annealing/extension temperature. This can help alleviate difficulties caused by secondary structures within RNA samples and/or primers.

In cases where inhibition of the reaction is possible, try reducing the sample amount¹ or add 0.4 – 4.4 mg/ml BSA to the reaction².

For more specific issues, please contact technical@pcrbio.com with the following information:

• Product size
• Reaction setup
• Cycling conditions
• Screenshots of amplification traces and melt profiles

1 Scipioni et al. A SYBR Green one-step real-time RT-PCR for detection of human and bovine noroviruses and control of inhibition. Journal of Virology.5:94 (2008). doi: 1186/1743-422X-5-94

2 Plante et al. Use of bovine serum albumin to enhance detection of enteric viruses by RT-qPCR from food washing concentrates. Letters in 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 per mL of 4x Lyo-Ready Probe Mix used. Due to the high concentration of the RTase, the provided volume is very small, so it is advisable to spin down the tube prior to each pipetting task.

While the 2 kit components are stable and with long shelf life (see expiry dates for each component), the 4x Lyo-Ready Probe 1-Step mix (obtained by adding 3200x UltraScript® RTase to 4x Lyo-Ready Probe Mix) should be used immediately or may be stored for up to 3 days at 4 °C. Therefore, it is advisable to prepare only the amount of 4x Lyo-Ready Probe 1-Step Mix necessary and return the remaining parts separately to -30 °C to -15 °C storage.

In the product's instructions, we provide a general lyophilisation protocol tested for 2 mL glass vials containing 500 μL of Lyo-Ready Probe 1-Step Mix. We have observed that lyophilisation is easier (i.e., shorter cycles, and cakes appear more uniform with no evidence of shrinkage) when the Lyo-Ready Probe 1-Step mixture is diluted to 1x or 2x (with oligonucleotides and water), so we suggest performing this dilution prior to running.

Depending on the lyophilised volume, the materials of the vials (i.e., glass vs plastic), and the lyophiliser used, the cycle may be optimized.

If cakes are not correctly formed after a cycle, we recommend repeating with a more conserving cycle, where primary drying is performed at -50 °C and time is lengthened to ensure correct cake formation. Secondary drying time can also be increased if necessary.

We recommend using 400-1000 nM for each primer. There is some flexibility around this recommended concentration; however, primer concentration should not be increased beyond this range as significant effects on enzyme performance may occur. Typically the probe concentration is half that of the amplification primers, but optimization and validation are necessary for each primer pair.