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OneStep RT-PCR Kit From Qiagen
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Full Product Review
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The QIAGEN OneStep RT-PCR Kit allows reverse transcription and PCR amplification to be performed sequentially in a single tube, referred to as a “one-step” reaction. The QIAGEN OneStep RT-PCR Enzyme Mix contains Omniscript and Sensiscript Reverse Transcriptases, designed to perform reverse transcriptions of RNA amounts from 1 pg to 2 ìg; the mix also contains HotStarTaq DNA Polymerase for performing PCR. To ensure the temporal separation of both processes, the reaction is incubated for 15 min at 95°C after the reverse transcription reaction; incubation at this temperature inactivates Omniscript and Sensiscript Reverse Transcriptases while it activates HotStarTaq DNA Polymerase. The kit also contains QIAGEN OneStep RT-PCR Buffer which has been optimized to ensure high efficiency of both reverse transcription and PCR in a one-step RT-PCR. A dNTP mix (10 ìM each) is also provided in the kit. Q-Solution is also present in the kit as an optional tool for improving
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Review Synopsis
Product: OneStep RT-PCR Kit From Qiagen
The Good: This RT-PCR kit is reliable and very convenient to use.
The Bad: Every kit always ends with a waste of leftover product
The Bottom Line: QIAGEN’s OneStep RT-PCR Kit is a very convenient and reliable kit to perform RT-PCR on any RNA |
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suboptimal RT-PCR. Lastly, the kit contains RNase-free water to top-off the RT-PCR reaction mix. The kit can be stored at -20°C for at least one year without detectable lost of efficiency.
I regularly use this kit to study the mRNA levels of specific genes in mammary carcinoma cells. The handbook recommends a RT-PCR reaction mix of 50 ìL, but experience shows that using a 25 ìL reaction volume is enough to get RT-PCR results as good as using 50 ìL in a majority of cases; the kit then provides enough reagents for 200 reactions instead of 100. However, 4% of Enzyme Mix in the final reaction mix is the minimum required to guarantee optimum reaction efficiency and reproducibility.
The convenience of the one-step reaction system allows setting up and performing series of reactions in a reasonably short period of time. It generally takes me 5 to 10 minutes to prepare the master mix. Total prep time (i.e. aliquoting the master mix and adding primers and RNA) varies according to the number of reactions being set up. I generally find that it takes me a total of 15 to 20 minutes to set up 10 RT-PCR reactions. The results of RT-PCR are consistent. I have performed several control RT-PCRs on the same quantity of the same RNA and have always obtained the same band intensities for these samples when run on an agarose gel.
The volumes of the various reagents included in the kit are not in accordance with the volumes of each product required to perform RT-PCR, resulting in some products being left over and eventually, wasted. Moreover, so far, I have not had to perform reactions with the Q-solution. As an optional tool, this Q-solution should be suggested, but not be included in the kit. In summary, the price of the kit includes the price of products that will never be used.
Author
Severine Brunet-Dunand
PhD Student
Molecular and Epigenetics Institute
The Liggins Institute
New Zealand |
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