RNA amplification is a process that takes a few copies of a section of ribonucleic acid (RNA) and creates thousands of copies that are identical in composition. Pieces of RNA are macromolecules, composed of strings of nucleotides, so amplification is a several-step process that involves the reproduction of the exact sequence of nucleotides from a piece of RNA. RNA amplification is useful in research involving viruses, whose genetic material can be stored as memory RNA (mRNA) instead of DNA; the study of gene expression in organisms; and the creation of proteins from transcriptional RNA (tRNA). To amplify RNA, a complimentary sequence is created and amplified, then used as a template for the creation of RNA copies. A DNA intermediary is needed for RNA amplification is because RNA generally occurs only in a single-strand, unpaired form, so a DNA template must be created for the RNA transcriptase enzyme to function; RNA cannot be transcribed from itself.
The RNA sequence of interest must first be isolated from contaminant material before RNA amplification can begin. After this is done, the enzyme reverse transcriptase is added to the RNA template. Reverse transcriptase is used to create a cellular DNA (cDNA) sequence of base pairs complementary to the original mRNA. This first strand of cDNA can then be used to create second strands of cDNA that can be used in polymerase chain reaction, a process that amplifies DNA strands through repeated cycles of DNA replication and subsequent separation of the two strands. The cDNA usually contains primers, or enzymes on one end of the DNA sequence, that serve as signals for nucleotide binding, and thus, the production of mRNA strands that are identical to the original sequence.
Several methods for RNA amplification exist, but the method that yields the largest amount of mRNA that is faithful to the original copy is called the template switching method. This method uses the reverse transcriptase enzyme from the Moloney murine leukemia virus, which adds a primer region to the end of the first-strand cDNA after creating this strand from the RNA template. This primer region is added to the second strand of cDNA as well, from which amplified RNA copies are made. Another enzyme, called a promoter, can be bound to the primer region on the second strand of cDNA, which drastically increases the amount of RNA copies that are produced from this cDNA strand through transcription.
