You can tell a lot about a cell by looking at its RNA, but these transitory molecules are difficult to study directly. RNA degrades easily because the extra hydroxyl group in the ribose sugar is highly reactive. DNA is more stable because it has a deoxyribose sugar backbone and double-stranded structure. That’s why scientists use reverse transcription to make complementary DNA (cDNA), which captures the RNA sequences in DNA form. Making cDNA is the first step in many molecular biology applications—from cloning and RT-PCR to microarrays and next-generation sequencing.
To maintain the validity of experimental results, you need cDNA that faithfully represents the template RNA. Major hurdles in cDNA synthesis reactions include the secondary RNA structures that may slow or even halt reverse transcription. Degradation of template RNA by the intrinsic RNAase activity of reverse transcriptases (RTs) is another problem that leads to truncated cDNA. Also, inhibitors present in RNA samples may reduce polymerization activity of the reverse transcriptase enzymes. Template degradation and inefficient polymerization result in cDNA of low quality and quantity.
To boost enzyme performance, scientists at Thermo Fisher Scientific used molecular evolution and rational design to engineer a better reverse transcriptase. Starting with the wild-type RT gene from the Moloney murine leukemia virus (M-MuLV), our scientists introduced a number of improvements to help you achieve better cDNA yields, longer products, and greater representation of input RNA.
Enzyme boost #1: Increased thermostability
Single-stranded RNA forms hairpin loops and other secondary structures, which can interfere with cDNA synthesis. The ability of a reverse transcriptase to tolerate high temperatures can help overcome this challenge. Wild-type M-MuLV RT works at 37–42°C, while our modified enzyme maintains activity up to 50–55°C. Elevated reaction temperatures destabilize RNA secondary structures, allowing the RT to read through the sequence, thus increasing cDNA yield.
Enzyme boost #2: Diminished RNase H activity
The first strand of cDNA synthesis creates DNA-RNA hybrid molecules. RTs often have built-in RNase H activity—the ability to hydrolyze RNA before completing the second cDNA strand. However, too much RNase activity can degrade template RNA prematurely, which can lower the yield and length of cDNA products. Our RT includes modifications that alter or significantly reduce RNase H activity resulting in an increase yield of full-length cDNA synthesis products.
Enzyme boost #3: Higher processivity
Substances that bind to RNA and interfere with cDNA synthesis are commonly carried over from RNA sample sources. Processivity is a key enzyme feature that refers to the number of nucleotides incorporated during a single binding event. Increased processivity correlates with tighter substrate binding and can improve resistance to these inhibitors. High processivity lets RTs synthesize longer cDNA strands in a shorter reaction time. For our improved RT, it takes just 15–30 minutes for cDNA synthesis depending on whether genomic DNA is removed or not. This efficiency brings higher yield and superior sensitivity with low quality or low quantity RNA samples, even RNA from single cells.
Before your next quantitative RT-PCR experiment or RNA sequencing analysis, make sure to avoid the biggest hurdles to reverse transcription. With increased thermostability, switched-off RNase H activity, and high processivity, our engineered RT enzyme was developed for optimal performance in cDNA synthesis. These three key improvements to the wild-type M-MuLV RT including increased thermostability, diminished RNaseH activity, and higher processivity, give you a robust tool to produce full-length cDNA that accurately represents your RNA of interest.
For Research Use Only. Not for use in diagnostic procedures.