cDNA synthesis from high GC templatesReverse transcription of RNA to complementary DNA (cDNA) is the first step in many molecular biology workflows including gene expression studies using real-time PCR, gene cloning and in the creation of cDNA libraries.
For accurate downstream analysis it’s essential that the reverse transcriptase used in this step generates cDNA products that are representative of the original RNA population, even with challenging samples, such as RNA with a high GC content and/or secondary structure. Efficient reverse transcription of these RNAs may exceed the capabilities of most reverse transcriptases. The enzyme may stop or dissociate from an RNA template at sites with complex secondary structure, or skip over looped-out regions, resulting in truncated cDNA or cDNA with internal deletions. If the RNA has a high GC content, the tight association of RNA–DNA hybrids can interfere with primer binding resulting in lower cDNA yields.
When working with difficult RNA samples such as these, we recommend the highly thermostable UltraScript 2.0 Reverse Transcriptase and cDNA Synthesis Kits. This range of products can be used with reaction temperatures of over 55°C which help to denature RNA with a high GC content or secondary structure, giving improved specificity, higher cDNA yields, and more full length cDNA product.
UltraScript 2.0 Reverse Transcriptase & Kits
Our high capacity, highly thermostable reverse transcriptase can be used with reaction temperatures of 55-65°C and above, designed to problem-solve the most difficult RNA samples.