Reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into DNA. The performance of reverse transcriptase will directly affect the final performance of molecular diagnostic reagents. In response to the pain points of molecular diagnostic reagent manufacturers, Yeasen has carried out the directional transformation on reverse transcriptase. Reverse transcriptase has excellent performance and is well received by customers!
Reverse transcriptase is an RNA-dependent DNA polymerase. It can transcribe the template RNA and forms complementary DNA (cDNA). Single-stranded cDNA is converted into double-stranded DNA using DNA polymerase. So reverse transcriptases are commonly used to quantify the level of mRNA synthesis when combined with the polymerase chain reaction technique, called RT-PCR. Quantitative RT-PCR assays are commonly used in disease diagnosis. Therefore, reverse transcriptase plays an important role in scientific research and practical application.
Common reverse transcriptases are M-MLV and AMV, among which M-MLV is better than AMV. But conventional RT enzymes have two active sites, one executing the polymerase activities and another executing the ribonuclease H activity. RNase H is an endonuclease able to degrade the RNA moiety of the DNA–RNA hybrids, family. Therefore, reverse transcriptase lacking RNase H activity is beneficial to cDNA synthesis. Besides that, using a thermostable version of RT is beneficial for lowering the nonspecific nucleic acid amplification and minimizing the impact of complex secondary structures.
Yeasen has carried out directional modification on reverse transcriptase, which removes RNase H activity, thereby improving the ability of cDNA synthesis. Using protein evolution technology to improve the thermostability of reverse transcription.
Yeasen's reverse transcriptase not only has strong synthesis ability but also a strong tolerance ability. In addition, Yeasen has strict requirements on quality. It is the best choice for reverse transcriptase.
4.1 Resistant to high temperature of 65°C
Using 500ng of 293T cell total RNA as a template, reverse transcription was performed at 42°C-65°C using Hifair™ Ⅲ Reverse Transcriptase. Take 1 μL of cDNA as a template to amplify the TFRC gene (4.4kb).
Figure 1. Perform reverse transcription at 42°C-65℃
4.2 Compatible with complex templates
The reaction temperature of 55°C can satisfy the reverse transcription of conventional templates and secondary structure templates, and the reaction temperature of high GC templates can be appropriately increased to 65°C.
Figure 2. Suitable for GC-rich templates and secondary structure templates
4.3 Excellent extension performance
Using 500ng total RNA of 293T cells as a template and Oligo(dT) as a primer, cDNA was synthesized using Hifair™ Ⅲ Reverse Transcriptase. Take 1 μL of cDNA as a template to amplify 500bp of 5' of the DY1C1N1 gene (19.9kb). The results showed that Hifair™ Ⅲ Reverse Transcriptase can synthesize cDNA up to 19.8kb.
Figure 3. Up to 19.8kb cDNA can be synthesized
4.4 Fast synthesis
Using the RNA of 293T cells as a template, reverse transcription was carried out according to the standard procedure in the manual, and the reverse transcription time was set according to 5, 10, 15, 30, 45, and 60 minutes respectively. Hifair™ III Reverse Transcriptase can synthesize a 6kb cDNA within 5 minutes, and its specificity and yield are superior to those of the V brand.
Figure 4. 6kb cDNA can be synthesized within 5 minutes
4.5 Wide linear range
Using 5pg-5μg total RNA of 293T cells as a template, cDNA was synthesized using Hifair™ III Reverse Transcriptase. 1 μL of cDNA was used as a template, and qPCR was performed on the four genes, and the results showed that the amplification efficiencies were all between 98% and 100.10%. It shows that cDNA can be efficiently synthesized in a wide linear range of templates.
Figure 5. cDNA can be synthesized efficiently in a wide template range
4.6 Strict quality control
To ensure the stable performance of Yeasen's reverse enzyme, Yeasen strictly implements the requirements in the production process to stabilize the production process and ensure batch stability. At the same time, quality inspection is strengthened to detect residues including exonuclease, nickase, RNase, etc., to ensure that there are no residues, so as not to affect downstream experiments.
Figure 6.Residue detection
The products provided by Yeasen are as follows.
Table 1. Related products
|Hifair™ Ⅲ Reverse Transcriptase (200 U/μL) (inquire)
|Hifair™ V Reverse Transcriptase (200 U/μL)
|Hifair™ III Reverse Transcriptase, 200 U/μl, Glycerol-free (inquire)
|Hifair™ V Reverse Transcriptase (inquire)
|Murine RNase Inhibitor
|Murine RNase Inhibitor (40 U/µL)
|Murine RNase Inhibitor (200 U/µL, Glycerol-free) (inquire)
|Hotstart Taq DNA Polymerase
|Hieff Unicon™ Hotstart Direct Taq DNA Polymerase,50 U/μL (inquire)
|Hieff UNICON™ Hotstart E-Taq DNA Polymerase, 5 U/μL