QuantiTect Reverse Transcription Kit
For fast cDNA synthesis enabling sensitive real-time two-step RT-PCR for gene expression analysis
For fast cDNA synthesis enabling sensitive real-time two-step RT-PCR for gene expression analysis
Cat. No. / ID: 205311
Using the QuantiTect Reverse Transcription Kit, contaminating genomic DNA in RNA samples is effectively and rapidly removed with the unique gDNA Wipeout Buffer (see figure Effective genomic DNA removal for accurate real-time RT-PCR). Elimination of genomic DNA is crucial for accurate gene expression results, and design of RNA-specific primers or probes is not always possible. With gDNA Wipeout Buffer, time is saved and costs are reduced, since a separate DNase digestion is unnecessary, either during or after purification of RNA samples.
The high RNA affinity of Quantiscript Reverse Transcriptase, in combination with Quantiscript RT Buffer, enables high yields of cDNA from any RNA template (see table “Higher cDNA yields for less abundant transcripts”). Even difficult templates, such as those with high GC-content or complex secondary structure, are successfully reverse transcribed.
|CT values for IL12A
|CT values for IL1RN
|Input RNA (ng)||QIAGEN||Supplier AII||QIAGEN||Supplier AII|
The RT Primer Mix contains a specially optimized mix of oligo-dT and random primers that enable cDNA synthesis from all regions of RNA transcripts, even from 5' regions (see figure Sensitive detection of a target at the 5' region of a 12.5 kb transcript). In contrast to kits from other suppliers, the QuantiTect Reverse Transcription Kit provides high yields of cDNA template for real-time PCR analysis regardless of where the amplified target region is located on the transcript, and provides greater sensitivity in the detection of low-abundance genes (see figure " Higher sensitivity in real-time, two-step RT-PCR"). The QuantiTect Reverse Transcription Kit also enables greater reproducibility in real-time RT-PCR.
QuantiTect Reverse Transcriptase is a novel blend of Omniscript and Sensiscript Reverse Transcriptases, which has a high affinity for RNA and is capable of cDNA synthesis from a wide range of RNA amounts (10 pg to 1 µg). In contrast to kits from other suppliers, the QuantiTect Reverse Transcription Kit provides high yields of cDNA template for real-time PCR analysis regardless of where the amplified target region is located on the transcript. Even difficult templates, such as those with high GC-content or complex secondary structure, are successfully reverse transcribed. QuantiTect RT Buffer has also been optimized to be compatible with real-time PCR buffer.
To obtain accurate results in real-time RT-PCR gene expression assays, it is important that only cDNA is amplified and detected. Interference by genomic DNA can be avoided by designing primers or probes that span an exon/exon boundary. However, in cases where this is not possible (e.g., the cDNA is from a single-exon gene), it is essential that the starting RNA sample is free of genomic DNA. Using the QuantiTect Reverse Transcription Kit, contaminating genomic DNA in RNA samples is effectively and rapidly removed with unique gDNA Wipeout Buffer. Time is saved and costs are reduced, since a separate DNase digestion not required, either during or after purification of RNA samples. Also, design of RNA-specific primers or probes is unnecessary.
|gDNA Wipeout Buffer||Detection of RNA only in real-time RT-PCR|
|Quantiscript Reverse Transcriptase||Use of a wide range of RNA amounts (10 pg to 1 µg RNA) |
|Quantiscript RT Buffer||Read-through of difficult templates|
|RT Primer Mix||cDNA synthesis from all regions of transcripts, even from 5' regions|
Genomic DNA removal and cDNA synthesis take only 20 minutes with the QuantiTect Reverse Transcription Kit (see flowchart " Fast and convenient cDNA synthesis"). The procedure is fast and convenient, since both reactions are run using the same incubation temperature and are set up using master mixes.
The QuantiTect Reverse Transcription Kit includes everything you need for fast cDNA synthesis. Purified RNA is briefly incubated in gDNA Wipeout Buffer to effectively remove contaminating genomic DNA. In contrast to other methods, the RNA sample is then used directly in reverse transcription, using a master mix prepared from Quantiscript Reverse Transcriptase, Quantiscript RT Buffer, and RT Primer Mix. With Quantiscript Reverse Transcriptase, RNA can be transcribed at low temperatures, even through complex 2° structure, ensuring that the RNA will stay intact — the entire reaction takes place at 42°C and is then inactivated at 95°C. Additional steps for RNA denaturation, primer annealing, and RNase H digestion are not necessary.
The QuantiTect Reverse Transcription Kit allows highly efficient and sensitive real-time RT-PCR for all types of starting material, including laser-microdissected samples and tissue biopsies.
Genomic DNA removal and cDNA synthesis take only 20 minutes with the QuantiTect Reverse Transcription Kit. The procedure is fast and convenient since both reactions are run using the same incubation temperature and are set up using master mixes. In contrast, the procedure for the kit from Supplier I is much longer and requires more "hands-on time" due to additional pipetting steps and frequent changes in incubation temperature.
|Applications||Quantification of (even low-abundance) transcripts|
|Sample/target type||RNA template|
|Enzyme activity||Reverse transcription|
|Real-time or endpoint||Real time|
|Reaction type||Two-step, cDNA production, genomic DNA digestion|
|Single or multiplex||Single|
Ensure PCR amplicons are as short as possible, ideally 60–150 bp. Always use the same algorithm or software to design the primers and probes. For optimal results, only combine assays that have been designed using the same parameters.
Check the functionality of each set of primers and probes in individual assays before combining the different sets in the multiplex assay. Choose compatible reporters and quenchers based on a specific instrument. See How do I select appropriate reporter and quencher combinations for multiplex PCR.
Yes, it is possible to use the QuantiTect Reverse Transcription Kit for bacteria. The RT Primer Mix provided in the kit is a unique, optimized blend of random primers and oligo-dT allowing high cDNA yields from all regions of RNA transcripts. It has successfully been tested for Reverse Transcription in bacteria as well. We strongly recommend to isolate bacterial RNA using the RNeasy Mini Kit prior to performing Reverse Transcription. This will ensure the high prep quality necessary for optimal RT results with the QuantiTect Reverse Transcription Kit.
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.
Although both are an optimized mixture of Omniscript and Sensiscript Reverse Transcriptases, the mixture provided in the QuantiTect Reverse Transription Kit is optimized for random priming in a two-step reaction, whereas the mixture in the QuantiTect Probe RT-PCR Kit is optimized for gene-specific priming in a one-step RT-PCR reaction.
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:
No, QuantiTect Primer Assays are supplied as lyophilized, premixed primer pairs. Reaction components for SYBR Green real-time RT-PCR must be purchased separately.
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.
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 (Tm) 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 Tm, but approximately 3ºC lower than the specific amplicon Tm.