QuantiFast Pathogen +IC Kit

Using the QuantiFast Pathogen + IC kits on the Rotor-Gene Q:

No. UNG treatment does not provide any advantage for the QuantiFast and Rotor-Gene PCR kits because the mastermixes do not contain dUTP. Use the QuantiTect kits if you intend to use the UNG treatment.

If the extra peaks seem irregular or noisy, do not occur in all samples, and occur at temperatures less than 70 ºC, then these peaks may not represent real PCR products and instead may represent artifacts caused by instrument settings.

Usually extra peaks caused by secondary products are smooth and regular, occur reproducibly in most samples, and occur at temperatures greater than 70 ºC. Characterization of the product by agarose gel electrophoresis is the best way to distinguish between these cases. If only one band appears by agarose gel then the extra peaks in the dissociation curve are instrument artifacts and not real products. If this is the case, refer to the thermal cycler user manual, and confirm that all instrument settings (smooth factor, etc.) are set to their optimal values.

The REST 2009 (Relative Expression Software Tool) software applies mathematic models that compensate for the different PCR efficiencies of the gene of interest and reference genes. In addition, the software can use multiple reference gene normalization to improve the reliability of result, as well as provides statistical information suitable for robust comparison of expression in groups of treated and untreated. QIAGEN offers the REST 2009 software free of charge.

Check the template quality and integrity by amplifying an endogenous control gene. Check the amplicon by QIAxcel Advanced system or agarose gel electrophoresis to show that amplification was successful.

Determine whether the gene of interest is expressed in your sample. See How can I find out if my gene of interest is express in a specific tissue type or cell line.  Ensure the assay setup and cycling conditions are correct, and that the data collection channel matches the emission wavelength of the fluorescent dye used. Use a control sample in which the gene of interest is definitely expressed.

If the issue persists, please send the original run file to QIAGEN Technical Services.

For duplex analysis, using non-fluorescent quenchers (e.g., Black Hole Quencher^®) is preferred over fluorescent quenchers (e.g., TAMRA fluorescent dye). For triplex and 4-plex analysis, QIAGEN strongly recommends using non-fluorescent quenchers. Generally, use the green channel, the yellow channel, and the orange and crimson channels to detect the least abundant target, the second least abundant target, and the two most abundant targets, respectively. For instrument-specific recommendations, please see the handbooks for the QuantiTect Multiplex PCR kit, QuantiFast Multiplex kit or Rotor-Gene Multiplex kit.

No, the QuantiFast Pathogen PCR +IC kits are designed for use with hydrolysis probes (also known as TaqMan® probes) only.

See trademarks. 

The gDNA wipeout buffer incubation step can be skipped when the total RNA is free from genomic DNA. However, the gDNA wipeout buffer is still required to be added because the reverse transcription step is optimized in the presence of components in the gDNA wipeout buffer.

Reliable HRM analysis results depend on template quality, highly specific HRM PCR kit with a saturation dye, a real-time instrument with HRM capability, and powerful software package. Factors critical for successful HRM analysis are:

• Use the same genomic DNA purification procedure for all samples being analyzed by HRM. This avoids variation due to differing composition of elution buffers.
• DNA template concentrations should be normalized using the same dilution buffer. Ensure the C_T values are below 30 and differ no more than 3 C_T values across individual samples.
• Design assays with amplicon length 70–350 bp. For SNP analysis, use amplicon length 70–150 bp.
• Always start with 0.7 µM primer concentration

For more details, please refer to the HRM Technology – FAQs and the Critical Success Factor for HRM performance.

Principally, yes. However, additional factors influencing the CT are:

1. Extraction efficiency, which depends on extraction method and sample material.

2. Volume of the eluate used for PCR.

Examples: Use of the Internal Control according to handbook instructions (0.1µl per 1µl elution volume) for two different workflows.

• Workflow 1: High extraction efficiency: use of 5 µl of the eluate for PCR results in a CT value of 29-30.
• Workflow 2: High extraction efficiency: use of 10 µl of the eluate for PCR results in a CT of 28-29.

Depending on the workflow, the amount of Internal Control to be added to the extraction might have to be adjusted to more than 0.1 µl per 1 µl elution volume, or to less than 0.1 µl per 1 µl elution volume, in order to achieve a CT value within the expected range.

Running the Internal Control DNA/RNA provided with the QuantiFast Pathogen +IC Kit in a separate reaction can serve as a reference for adjusting the amount of Internal Control DNA/RNA (High conc.) to be added to the extraction.

Yes, the Internal Control RNA (High conc.) and Internal Control DNA (High conc.) templates are available under a separate catalog number. After reconstitution according to the description in the handbook, these IC templates have a 10x higher concentration than the Internal Control templates provided in the kits.   They are sufficiently concentrated to be spiked into the sample prep without replacing too much of the sample input volume.

**Please note that there is no assay (primer/probe set) for amplification of the IC included in these separate catalog numbers. The assay is only included in the QuantiFast Pathogen PCR +IC Kit and QuantiFast Pathogen RT-PCR +IC Kit.  The ICs cannot be used with the QuantiTect Virus kits because the assay (primer/probe set) for the detection of the IC is provided with the QuantiFast Pathogen Kits only.

After reconstitution according to handbook instructions, the Internal Control (IC) templates provided with the kits have a ready-to-use concentration for direct use as an amplification control in PCR or RT-PCR. For each 25 µl reaction, 2.5 µl of the IC are added, resulting in an expected CT value of approximately 29-30.

In order to achieve the same CT value when using the IC as an extraction control, more IC template has to be spiked in before extraction. The exact amount depends mainly on the elution volume. For each 1 µl of elution volume, 0.1 µl of the IC High conc. should be added to each of the extraction samples. This should result in a CT value in the PCR or RT-PCR reactions of approximately 29-30.

Examples: For 100 µl of elution volume, 10 µl of the IC, per sample, should be added to the lysis buffer or lysate. For 50 µl of elution volume, 5 µl of the IC, per sample, should be added to the lysis buffer or lysate.

Depending on primer design and copy number of target, primer-dimer may occur and its signal might be detected. Typical strategies against this are to optimize PCR conditions and/or redesign the assay.

Alternatively, an additional data-acquisition step can be added to the 3-step cycling protocol. First, determine the melting temperatures (T_m) for both the amplicon and the primer-dimer. Then, add a 15 second data-acquisition step with a temperature that is higher than the primer-dimer T_m, but approximately 3ºC lower than the specific amplicon T_m.