FastGene® Scriptase II
Engineered reverse transcriptases allowed the synthesis of cDNA from very low amounts of RNA. Mutations are inserted into the RNase H domain of the MuLV‘s reverse transcriptase. Therefore, by not degrading the RNA during the first-strand synthesis, a higher yield of full-length cDNA is obtained. Additionally, a higher thermal stability increases the robustness of the enzyme. The FastGene® Scriptase II is exactly one of those engineered enzymes. With its mutation in the RNase H domain and higher thermal stability, it is the optimal choice for more complex applications, such as RT-qPCR and NGS.
- Quantification of Gene Expression
- Next Generation Sequencing
- Low RNA concentration
- Difficult templates
Lower RNase H activity for longer cDNA
The FastGene® Scriptase II has a modified RNase H domain. The RNA is therefore not degraded and serves as a template for longer cDNAs, resulting in fragment size of up to 12 kBp.
Engineered enzymes – optimized for qPCR
The FastGene® Scriptase II delivers superior cDNA templates for downstream applications, e.g. qPCR and NGS. The resulting full-length cDNA gives a complete picture of the gene and is able to show modification, e.g. splicing variants.
Comparison of different PCR results
Fig. 1: Comparison of multiplex PCR using cDNA produced by Competitor I‘s SS-II enzyme and FastGene® Scriptase II at 42 °C and 50 °C.
Fig. 2: Comparison of qPCR results using primers for GAPDH and cDNA produced by using different RNA starting concentration by Competitor I‘s SS-II enzyme and FastGene® Scriptase II at 42 °C.
Fig. 3: Comparison of qPCR results using primers for YWHAZ and cDNA produced by using different RNA starting concentration by Competitor I‘s SS-II enzyme and FastGene® Scriptase II at 42 °C.
Comparative study of reverse transcriptase reaction using RNA extracted from zebrafish fertilized eggs
Cloning of isoflavone biosynthesis related enzyme using RNA extracted from soybean
Very fast reverse transcription reactions
Comparative study of reverse transcriptase reaction using RNA extracted from peritoneal cells of sterile peritonitis model mouse
Comparative study of RT-reaction by using RNA extracted from mouse lymph node
Reverse transcriptase reaction for quantitative expression analysis using RNA extracted from mesenteric adipose rat tissue
The step 5 min 65°C in the protocol is not mentioned in the manual of Scriptase II ReadyMix. What is the purpose of this step and can I skip it if I only use the single enzyme Scriptase II?
The 65°C step is to avoid secondary structure of RNA molecules. RNA is a single stranded molecule which forms hairpin structure with itself to achieve better stability. The step is a kind of safety step but in many cases, it is not necessary. So, you can eliminate this step if you get good results in both cases.
What is the optimal temperature range for Scriptase Basic and Scriptase II?
the optimal temperature range for the Scriptases is 42°C to 50°C. A temperature above 50°C is not recommended.
Can I use the Scriptase II with a high amount of RNA (2.5 µg per 20 µl)?
We recommend a max of 1 µg. Reason: the highly expressed genes will over proportionally be represented in such high concentration. Meaning the chances of finding a high-concentrated mRNA is much higher than a low concentrated one. If you stick to 1 µg, the amount of enzyme to mRNA will be much higher, guaranteeing the complete RT of all mRNA.