The successes and future prospects of the linear antisense RNA amplification methodology

摘要

In the first round, first-strand cDNA synthesis by reverse transcription (RT) is primed from mRNA after oligo(dT)-T7 primer anneals to the poly(A) tail of mRNA. RNase H is then used to digest portions of the bound mRNA to create RNA fragments that serve to prime second-strand cDNA by DNA polymerase (pol). Finally, aRNA is amplified via linear transcription by T7 RNA polymerase, using the T7 RNA polymerase promoter incorporated in the double-stranded cDNA. In the second round, first-strand synthesis is primed by random primers instead of the oligo(dT)-T7 primer by reverse transcriptase using the aRNA as a template instead of mRNA. After RNA denaturation, second-strand synthesis is primed with the oligo(dT)-T7 primer, which binds to the poly(A) tail of the cDNA created during first-strand synthesis by DNA polymerase. Finally, RNA is again linearly amplified through the enzymatic activity of T7 RNA polymerase acting on its promoter that is incorporated into the double-stranded cDNA2,3. Credit: Marina Spence/Springer Nature.