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Codex® HiTemp Reverse Transcriptase is a highly sensitive and robust enzyme specifically engineered for thermostability and performance in challenging conditions. This RNase H-minus enzyme is automation-friendly with extended stability at ambient temperatures. An expedited protocol saves considerable time to results while retaining key performance metrics such as sensitivity, making it the ideal choice for custom one-step RT-qPCR assays.
Codex® HiTemp Reverse Transcriptase can perform at temperatures as high as 70 °C across multiple gene targets.
A one-step RT-qPCR mix containing Codex® HiTemp Reverse Transcriptase and wild-type Taq polymerase was challenged by performing reverse transcription at 50 °C, 60 °C, and 70 °C, for a panel of 30 unique genes. Codex® HiTemp Reverse Transcriptase was able to reliably detect all targets at each temperature tested.
Codex® HiTemp Reverse Transcriptase demonstrates high sensitivity in a 5-minute RT reaction.
Sensitivity and efficiency of a one-step RT-qPCR mix containing Codex® HiTemp Reverse Transcriptase and wild-type Taq polymerase was compared between a standard and expedited protocol across RNA inputs ranging from 100 ng to 0.1 pg. Reverse transcription was carried out in either 15 or 5 minutes, while the annealing-extension step during amplification was either 30 or 15 seconds per cycle respectively. Cq values were similar for both for each input amount. The expedited protocol retained sensitivity down to 0.1 pg input RNA.
Codex® HiTemp Reverse Transcriptase maintains performance with degraded RNA.
The performance of a one-step RT-qPCR mix containing Codex® HiTemp Reverse Transcriptase and wild-type Taq polymerase was benchmarked against a commercially available one-step RT-qPCR mix (Supplier 1) using RNA with varying degrees of degradation as input. Both one-step RT-qPCR mixes performed similarly for intact and minimally degraded RNA samples (RQS values of 9.1 and 7.2, respectively). However, the Codex® HiTemp Reverse Transcriptase containing mix demonstrated improved performance over Supplier 1 when RNA samples with higher degrees of degradation were used. Here, the ΔCq values between intact and highly degraded RNA were as low as 0.54 cycles for the one-step RT-qPCR mix containing Codex® qPCR Reverse Transcriptase, compared to 0.90 cycles for Supplier 1.
