さらに優れたパフォーマンスを実現するために、ezDNase付きSuperScript IV VILOマスターミックスも提供しています。ezDNaseは、非常に簡素化されたゲノムDNA除去ステップを通じて、RT-qPCRワークフローをさらに加速します。これにより、逆転写プロトコル全体の時間が劇的に短縮され、従来のDNase処理中のRNAの損失または損傷による遺伝子発現の変動が減少します。
In your SuperScript IV RT protocols, there is no ezDNase inactivation step. Will active ezDNase affect RNA or the RT reaction?
The Invitrogen ezDNase Enzyme is a novel DNase that is highly specific for double-stranded DNA. It has no activity on single-stranded DNA in RT reactions (primers or probes), or on RNA. The enzyme is also thermolabile—it is inactivated quickly at temperatures typical for the SuperScript IV RT reaction (e.g., 50°C). The additional inactivation step is therefore not required in RT-qPCR applications.
Which SuperScript IV RT format do you recommend for real-time PCR applications?
For RT-qPCR applications we recommend using the Invitrogen SuperScript IV VILO Master Mix (Cat. No. 11756050). The cDNA synthesis reaction setup with this master mix requires fewer pipetting steps and therefore reduces variation in the data. SuperScript IV RT, as a component of the master mix, offers the highest efficiency of cDNA synthesis step compared to competitors’ products.
Are there any significant changes in the SuperScript IV RT protocol compared to the SuperScript III RT protocol?
The only change is that the incubation time for the reverse transcription reaction has been reduced from 50 minutes to 10 minutes. All the other parameters and steps are the same.
Can I get comparable cDNA yield and length using the SuperScript IV RT 10-minute protocol as when using the 50-minute protocol for SuperScript III RT?
When compared with SuperScript III RT (and other manufacturers’ RTs) in a synthesis reaction for a 9 kb cDNA, SuperScript IV RT performed successful synthesis in just 10 minutes and did so with comparable (or improved) yield (as shown by gel band density).