Invitrogen’s Superscript™ III reverse transcriptase (RT) provides a fast, easy-to-use system for the generation of first strand cDNA (which can be used in subsequent PCR reactions) from mRNA. Gene-specific RT-PCR provides an excellent surrogate measure for comparing levels of message transcript (mRNA) in cells extracted from different tissues, cell lines, or cells exposed to different treatments, as mRNA levels cannot be accurately compared directly. We routinely use Superscript™ III in conjunction with Promega’s random hexamers (we find it is the most efficient way to amplify total RNA without generating bias), to generate high-quality, stable first-strand cDNA transcripts from mRNA extracted from fresh mouse and zebrafish tissues, human and mouse cell lines in culture, and even on RNA extracted from formalin-fixed paraffin-embedded (FFPE) brain and colon tissue, which speaks to the robustness of this product.
The procedure for use is quite simple. We normally synthesise first-strand template from 1-4 ug of total RNA, which (depending on the type of source cell/tissue type) comprises ~10-400 ng of mRNA. By combining random hexamers and deoxynucleotide triphosphates (dNTPs) with template RNA, heating to 65ºC then cooling rapidly, secondary RNA structure is denatured, allowing the hexamers to bind to random sequences on the RNA, thus, providing a priming template for subsequent cDNA synthesis. We then add the Superscript™ III enzyme (0.4 ul per reaction gives comparably high yields to the 1 ul suggested by the manufacturer, effectively more than doubling the number of reactions from one tube) and incubate at 50ºC for 60-90 minutes. This optimal reaction temperature differs from Superscript™ III’s predecessor, Superscript™ II, which showed optimal activity at 42ºC. This is the main change between the two Invitrogen products, and according to the manufacturer, this increased temperature leads to more efficient reverse-transcription due to the reduced presence of RNA secondary structure. Additionally, as the half-life of Superscript™ III is ~220 minutes at 50ºC, cDNA yields are far superior to that obtained with Superscript™ II and other RT’s (as claimed by Invitrogen).
In summary, Invitrogen’s Superscript™ III is the best RT, of the three I have used. The cDNA, once synthesized, can be stored at 4ºC with no appreciable degradation, although storage at -20ºC is recommended for longer periods. In my experience, using Superscript™ III has never once failed to generate good quality cDNA, even from difficult starting RNA templates (e.g. FFPE tissue, single zebrafish embryos). I would definitely recommend this product to anyone wishing to use reverse transcription for any application.
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on Apr 26, 2008 RamzesScript said:
Super Script III from Invitrogen is certainly no better than competition. My friend tested multiple enzymes on more demanding RNA template and Invitrogen Super Script III was one of the worst performing enzymes. Also Invitrogen ThermoScribe was not as good as you might expect. Increased temperature to 50 or to 55 did not help. Strong stops depending on secondary structure of RNA template were unacceptable for Invitrogen brand...
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Sebastian Dworkin
Graduate Student
Research Department
Peter MacCallum Cancer Centre
Australia
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