QuantiTect Reverse Transcription Kit

Yes, you can substitute the RT Primer Mix supplied in the FastLane Cell cDNA Kit and the QuantiTect Reverse Transcription Kit with your gene-specific primers. We suggest optimizinig the primer concentration by titration, starting at 1 uM, and gradually decreasing it to 0.5 uM final concentration in the reaction. Optimal amounts will depend on the specific primers you are using.

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.

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.

No, QuantiTect Primer Assays are supplied as lyophilized, premixed primer pairs. Reaction components for SYBR Green real-time RT-PCR must be purchased separately.

• For two-step RT-PCR, the QuantiTect Reverse Transcription Kit and QuantiTect SYBR Green PCR Kit are recommended
• For one-step RT-PCR, the QuantiTect SYBR Green RT-PCR Kit is recommended

To find a QuantiTect Primer Assay for your target gene of interest, please visit our GeneGlobe data base.

Shorter amplification products facilitate high PCR efficiencies. Ideally, amplicon length should be less than 150 bp for optimal amplification efficiency. PCR efficiencies close to 100% are a crucial prerequisite for accurate quantification of target copy numbers in real-time PCR.

The QuantiTect Reverse Transription Kit should be stored at -20°C immediately upon receipt. We recommend to aliquot the RT Primer Mix and keep it at -20°C.

If the kit is being used routinely, it may be convenient to prepare a premix of RT Primer Mix and 5x Quantiscript RT Buffer at a ratio of 1:4. Aliquot the premix and store at -20°C.

No, the T-Script® enzyme of the QuantiTect Whole Transcriptome Kit is an optimized blend for whole transcriptome amplification and cannot be substituted by Quantiscript Reverse Transcriptase of the QuantiTect Reverse Transcription Kit, or any other reverse-transcription enzyme.

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.