A multistep process involving the formation of double-stranded DNA (dsDNA) from single-stranded RNA (ssRNA) in the presence of the enzyme reverse transcriptase. The enzyme exhibits RNase H and DNA polymerase activity, both of which are essential for the reaction to complete.

Reverse transcription (PCR) helps detect gene expression at lower levels within cells and tissues. The reverse transcription method is divided into several steps. Each step is essential to ensure proper cDNA production and its effective use in future research.

RNA template preparation and purification: Reverse transcription begins with the isolation and purification of RNA from biological samples. This step must be handled carefully to avoid contamination and degradation, as poor RNA quality can lead to defective cDNA synthesis.

Primer binding to deoxyribonucleic acid (RNA): RNA primers are short RNA sequences that bind to specific parts of the RNA template during reverse transcription, allowing reverse transcriptase to initiate complementary RNA (cDNA) synthesis. These primers are short nucleotide sequences of RNA or DNA that ensure proper binding and initiation of cDNA synthesis.

For example, oligo(dT) primers bind to the poly-A tail of mRNA. However, random or gene-specific primers can be used in other situations, and the effectiveness and outcome of reverse transcription are directly influenced by the primer used.

Synthesis of first-strand complementary RNA by reverse transcriptase: Reverse transcriptase catalyzes the production of complementary RNA by detecting the RNA template and producing the corresponding RNA strand. This step reverses the traditional transcription process, which converts DNA to DNA. Reverse transcriptase is typically produced by retroviruses; however, modified versions are available for various experimental purposes.

RNA degradation and second-strand synthesis: RNase H activity degrades the RNA template using various techniques after the first strand of complementary DNA (cDNA) is produced. Double-stranded complementary DNA is produced when the second strand of DNA is generated.

This step is not always required, but when double-stranded cDNA is needed for cloning or PCR, it is essential to produce a complete cDNA product.

Companional cDNA amplification (when combined with PCR): After the complementary DNA is synthesized from the RNA template, the resulting complementary DNA can be amplified using PCR. This amplification is particularly useful for gene expression analysis and sequencing, as it enables genes to be studied starting from tiny amounts of RNA, allowing for more detailed studies.