Lower RNase H activity
Tolerance of highest temperature through bioengineering
High quality cDNA yields
Random hexamers and oligo-dT included
The additional hydroxyl group present in RNA facilitates the formation of hydrogen bonds. Hence, RNA is able to form very complex secondary structures. These secondary structures have a major influence on the efficiency of reverse transcriptases1,2 . The temperature of the reaction can be increased so that secondary structures are avoided. Nonetheless, majority of the wildtype and engineered enzymes is not able to tolerate hot temperatures.
FastGene® 55-Scriptase is an engineered reverse transcriptase optimized to tolerate higher temperatures (up to 60 °C). Its optimal enzymatic activity is achieved at 55°C. This leads to higher fidelity cDNA, as the secondary structure is reduced. The result is the best cDNA to be used in downstream applications such as qPCR. Additionally, the decision of using random hexamers or oligo-dTs is yours as both are delivered with the kit.
The avoidance of the secondary structure together with the reduced RNase H activity inherited from the MMuLV-reverse transcriptase enables the production of cDNA templates larger than 20 kBp. The larger cDNA templates give you a more accurate picture of the mRNA compared to smaller fragments, where transcripts can be left out due to the smaller fragments.
1. Brooks, E. M., Sheflin, L. G., & Spaulding, S. W. (1995). Secondary structure in the 3’ UTR of EGF and the choice of reverse transcriptases affect the detection of message diversity by RT-PCR. BioTechniques, 19(5), 806–812,814–815.
2. Wei, S., & To, S. S. T. (2015). Influence of RNA secondary structure on HEV gene amplification using reverse-transcription and nested polymerase chain reaction. Journal of Clinical Virology, 27(2), 152–161. doi:10.1016/S1386-6532(02)00170-1
The FastGene® 55-Scriptase outperforms majority of the competitors in yield. The yield of the cDNA was analysed using a very large product of 14905 bp. The reverse transcription reaction of 500 ng of total RNA was performed at four different temperatures. The competitors are:
The product was analysed using a 0.8 % agarose gel. The results can be seen in Figure 1:
|Figure 1 Comparing different reverse transcriptases. 500 ng of total RNA were used to reverse transcribe into cDNA and amplify a 14905 bp product. The reverse transcription was performed at 42°C, 50°C, 55°C & 60°C. The first lane shows wildtype MMuLV, followed by the FastGene® 55-Scriptase. The lane three and four show the reverse transcription of competitor engineered RTases. Lane 5 shows the amplification of a reverse transcriptase of competitor L.|
The yield of all polymerases is comparable at 42 °C. Nonetheless, it has been shown in the past that secondary structures are still present at 42 °C. Hence, the RTase must be able to tolerate higher temperatures. At 50°C, the wildtype MMuLV and the RTase from competitor L show considerably less product, when compared to the engineered RTases. The reduction of yield is also seen at 55 °C for the engineered RTases of Competitor I. The robustness of FastGene 55-Scriptase is shown at 60 °C, where it has the highest yield. The wildtype MMuLV and competitor L failed completely at 60 °C.
RNA is very fragil and isolation of low concentration of RNA is very common. Low concentrations are difficult to be reverse transcribed into cDNA for the majority of RTases. The sensitivity, high temperature and robustness of the FastGene® 55-Scriptase enable the amplification of very low amounts of RNA, as seen by the figure below:
Figure 2. Low concentrations of RNA as starting material.
Figure 3: Higher temperature during the reverse transcription leads to earlier CT-values.
The figure shows that the temperature near 55°C leads to a considerably lower CT-value when compared to 37°C or 42°C which are normally used for reverse transcription. This could be the difference between a false negative and a true negative result.
The amplification of complete genes is essential to study gene splicing. For very long genes it was a problem because majority of the RTases are able to create cDNA templates smaller than 13 kBp. FastGene® 55-Scriptase is able to create libraries of 20 kBp allowing a more complete picture of the gene expression. The complete picture is of course also important for normal-sized genes. The higher temperature also garantees less influence of RNA secondary structures to the created cDNA:
Fig.1: Comparison between cDNA produced by 1. FastGene® 55-Scriptase and 2. Competitor T. Competitor T was not able to produce a cDNA template for a large amplicon (14905 bp) in conditions avoiding secondary structures.
Multiplex PCR is a powerful assay enabling the detection of gene expression of multiple genes simultaneously. The advantages are that there is no variation in the starting material. All amplifications are done using the same template. Hence, the quality of the cDNA must be the highest to deliver a template for all primers in the reaction. As shown previously, the temperature at which the reverse transcription is performed can have a major impact on the quality of the cDNA (see section “Best in class, Consequences for the qPCR”).
We analyzed a Multiplex PCR using three different genes. The reverse transcription was performed at 3 different temperatures. The results are shown below:
Figure 2: Multiplex PCR using cDNA reverse transcribed by different RTases. Lane 4 shows the product of a multiplex PCR using FastGene® 55-Scriptase. Lanes 1 & 2 show engineered RTases from competitor I, lanes 3 and 5 wildtype MMuLV from competitor P and L, resp..
As shown in Figure 2, all reverse transcriptases were able to produce the three bands at 42 °C. As discussed before, the most secondary structures are still present at 42°C. Hence the analysis was performed at 55 °C as well. Two RTases out of three completely failed and at 60 °C, FastGene® 55-Scriptase was one of the two still being able to provide a complete cDNA.
The robustness of our engineered FastGene® 55-Scriptase was proven by incubating the enzyme at 50 °C for several hours. Afterwards the enzyme was used to reverse transcribe cDNA. This cDNA was then analyzed using endpoint PCR:
Figure 1: Results of an endpoint PCR using cDNA reverse transcribed using FastGene® 55-Scriptase incubated at 50 °C for 0, 1, 2, 4 and 8 hours.
The incubation of 8 hours at 50°C did not cause a significant change in yield. Nonetheless the band seemed less intense than without the incubation. Hence, a quantitative PCR assay was performed:
Figure 2: Results of an qPCR using cDNA reverse transcribed using FastGene® 55-Scriptase incubated at 50 °C for 0, 1, 2, 4 and 8 hours.
As seen in the figure, the difference of the threshold of the unincubated enzyme and of the enzyme incubated for 8 hours at 50 °C was only half a cycle. This could be due to loading variability rather than quality of the RNA.
The FastGene® 55-Scriptase is a very stable and robust enzyme able to tolerate elevated temperatures for a prolonged time without loss of enzyme activity.
|cDNA Synthesis kit||Manual for the cDNA Synthesis kit||Download|
|Reverse Transcriptase||Manual for the reverse transcriptase||Download|
|LS51||FastGene® 55-Scriptase Enzyme|| |
10 000 Units FastGene® 55-Scriptase Enzyme (200 Units/µl)
|LS61||FastGene® 55-Scriptase cDNA Synthesis kit|| |
10 000 Units FastGene® 55-Scriptase Enzyme (200 Units/µl)