qScript Ultra Flex Kit
- Enhanced Thermostability – Engineered for RT activity up to 65°C, overcoming challenging secondary structures
- Superior Speed & Ease of Use – 10 minute reaction time with fewer pipetting steps
- Maximum Yield & Sensitivity – Improved yields with limiting or compromised samples
- Ultimate Inhibitor Resistance – Overcome a wide array of PCR inhibitors (salt, heparin, hematin, etc.)
- Longer Transcripts – Ability to reverse transcribe single targets up to 20 kb
qScript Ultra Flex Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.
Description
qScript Ultra Flex Kit is easy-to-use and highly efficient for the synthesis of first-strand cDNA with flexible priming methods including oligo-dT, random primers and gene specific priming to reverse transcribe RNA to cDNA. A key component is a novel, state-of-the-art, RNase H deficient reverse transcriptase engineered for improved thermostability, velocity, processivity, and resistance to many common reaction inhibitors. qScript Ultra Flex Kit contains all required components for first-strand cDNA synthesis except RNA template and gene specific primers. The cDNA product is compatible with downstream 2-step RT-qPCR or RT-PCR procedures.
Details
|
Components |
95215-025 |
95215-100 |
|
5x qScript Ultra Reaction Mix Optimized master mix containing buffer, magnesium, dNTPs and qScript Ultra RT |
1 x 100 µL |
1 x 400 µL |
|
Oligo(dT) primers 10x concentrated in Enhancer solution |
1 x 50 µL |
1 x 200 µL |
|
Random primers 10x concentrated in Enhancer solution |
1 x 50 µL |
1 x 200 µL |
|
GSP Enhancer (10x) |
1 x 50 µL |
1 x 200 µL |
|
Nuclease-free Water |
1 x 1.5 mL |
2 x 1.5 mL |
Performance Data
Speed Comparison vs. Alternative Kits
Reaction Time Comparison.
Incorporating the highly rapid and processive qScript Ultra reverse transcriptase, the qScript Ultra Flex Kit requires only 10 minutes for first-strand cDNA synthesis over a broad range of input RNA quantities, even for very long cDNA products. Total reaction time including heat inactivation is only 15 minutes.
Reverse Transcription at Elevated Temperature
Thermostability and Activity at High Temperatures.
cDNA was synthesized in reactions using 5 ng total human RNA and a stable LDHA genespecific reverse primer. Each were incubated at the indicated temperatures, followed by reaction termination with products detected by qPCR. The lower Cq values for the engineered qScript Ultra reverse transcriptase indicate more efficient cDNA synthesis at high temperatures up to 65°C, with no loss in performance up to 60°C, critical for progressing through high GC-regions and regions of RNA secondary structure.
Comparison of yield
Yield Comparison.
In randomer-primed first-strand cDNA synthesis reactions, the qScript Ultra Flex Kit improves RNA conversion and allows for detection of sequences from highly degraded RNA isolated from FFPE liver tissue.
Sensitivity Range
Sensitivity Range.
Even with small quantities of highly degraded RNA extracted from FFPE liver tissue, qScript Ultra Flex Kit maintains linearity in cDNA synthesis over a range of RNA input and allows for a reproducible low limit of detection.
qScript Ultra Flex Dynamic Range
Dynamic Range.
In an oligo(dT)-primed, first strand cDNA synthesis reaction series, qScript Ultra Flex Kit enables a reproducible low limit of detection with a broad linear dynamic range over eight orders of magnitude.
Inhibitor Resistance Across and Wide Range of Sample Types
Inhibitor Resistance Comparison.
The robust, engineered qScript Ultra reverse transcriptase is tolerant to many common reaction inhibitors. In standard random-primed first-strand cDNA synthesis reactions, the qScript Ultra Flex Kit maintains high yields of cDNA products in the presence of several inhibitors as indicated by the lack of large qPCR Cq shifts in the treated samples. Alternative kits were tested with and without inhibitors according to the manufacture’s recommended protocol.
Long Range Capabilities
Long Range Capability.
cDNA synthesis reactions were carried out to 55°C for 10 minutes, and the cDNA used directly (-H) or treated with RNase H (+H). A portion of the products amplified using repliQa HiFi ToughMix. The Thermo Maxima reverse transcription mix has RNase H+ activity.
A: Various quantities of total RNA from rat brain were used for GSP-primed first-strand cDNA synthesis, then a 12.5 kb portion of the rat Dynein mRNA was amplified and analyzed. PCR amplification using repliQa HiFi ToughMix took only 54 minutes.
B: Various quantities of human total RNA were used for GSP-primed first-strand cDNA synthesis, then a 17.7 kb portion of the human SYNE1 mRNA was amplified and analyzed. PCR amplification using repliQa HiFi ToughMix took only 1 hour and 24 minutes.