Which step is necessary in RT-PCR to enable amplification of RNA-derived templates?

• A. Ligation of adapters to DNA
• B. Denaturation of double-stranded DNA
• C. Polymerase chain reaction amplification without prior conversion
• D. Reverse transcription converts RNA into cDNA so it can be amplified by DNA polymerase.

Answer: (D)

In RT-PCR, RNA cannot be directly amplified by DNA polymerase, so the essential step is reverse transcription: an enzyme uses the RNA as a template to synthesize complementary DNA (cDNA). That cDNA then serves as the template for standard PCR amplification by DNA polymerase. Without this conversion, there’s no DNA template for amplification. Ligation of adapters is related to sequencing library prep, not RT-PCR. Denaturation of double-stranded DNA is part of the PCR process itself but only after RNA has been converted to cDNA. Amplifying RNA directly with DNA polymerase isn’t possible because the template is RNA, not DNA, so the conversion to cDNA is the necessary prerequisite. In many protocols this happens in one tube (one-step RT-PCR), but the key step remains transforming RNA into cDNA before amplification.