Many biological processes, such as gene cloning and gene expression analysis using real-time polymerase chain reaction (qPCR) and high-throughput single cell transcriptome sequencing, require the use of high-quality complementary DNA (cDNA), which is transcribed from a specific mRNA using the enzyme reverse transcriptase. The type of reverse transcriptase and primer are significant in determining synthesis rate and reaction fidelity. The main processes in reverse transcription reactions are primer annealing, DNA polymerization, and enzyme deactivation. Moreover, the temperature and duration of these stages vary based on the primer used, the target RNA, and the reverse transcriptase employed. The kind of reverse transcriptase is significant since different enzymes have varying functional activity and capabilities. Reverse transcriptases, in general, feature an RNA H domain with endoribonuclease activity that employs an RNA template and a short primer complementary to the 3′ end of the RNA to direct the synthesis of first strand cDNA, which can then be used directly as a template for qPCR.
Depending on the RNA template and the downstream application, three different types of primers—oligo(dT) primers, random primers, and gene-specific primers—are employed to start reverse transcription. To avoid an abundance of truncated cDNA transcripts and a disproportion of cDNA generated from the 5′ or 3′ ends of the RNA, careful priming is required.
Overall, complete gene expression investigations are undertaken on a regular basis at Visikol, typically examining variation in expression generated by the application of a pharmacological compound, offering light on the potential mechanism of toxicity of potential compound candidates. Additionally, Visikol provides advanced drug discovery solutions such as 3D cell culture assays and tissue imaging high content screening, AI solutions for histological analysis of tissue sections, and much more. To learn more about Visikol’s services, please contact us.