1. Reverse Transcription (RT): RNA is converted into complementary DNA (cDNA) by an enzyme called reverse transcriptase.

2. Polymerase Chain Reaction (PCR): The cDNA produced from the reverse transcription step is subsequently amplified using PCR.

In RT-PCR, there are two main approaches: one-step and two-step. Each has its advantages depending on the experiment’s goals, RNA quality, and sample quantity.

In a one-step RT-PCR, both reverse transcription (cDNA synthesis) and PCR amplification are performed in the same tube without transferring materials.

• Simplicity: Fewer handling steps reduce contamination risks and save time.

• Speed: Fast and convenient, especially for high-throughput or diagnostic applications.

• Consistency: Since all reagents are in one tube, it minimizes variability between samples.

In a two-step RT-PCR, reverse transcription and PCR amplification are conducted separately. cDNA is synthesized first and can be stored or used for multiple PCR reactions.

• Flexibility: Allows using the same cDNA for multiple targets, making it ideal for studies with multiple genes.

• Sensitivity: Often more sensitive for low-abundance targets, as different primers can be used in each PCR reaction.

• Storage and Reuse: cDNA can be stored and reused, beneficial when sample availability is limited.

• Use one-step RT-PCR if you need a streamlined, rapid process, typically in high-throughput diagnostics.

• Opt for two-step RT-PCR for more complex or sensitive experiments that require flexibility and multiple analyses from the same sample.

The principle of RT-PCR (Reverse Transcription Polymerase Chain Reaction) involves two main steps:

• RNA Conversion: This step converts RNA into complementary DNA (cDNA) using an enzyme called reverse transcriptase.

• Primer Binding: A short piece of DNA called a primer binds to a specific sequence on the RNA molecule. This provides a starting point for the reverse transcriptase to begin copying the RNA.

• cDNA Synthesis: The reverse transcriptase enzyme adds nucleotides to the growing cDNA strand, complementary to the RNA template.

• Exponential Amplification: This step exponentially amplifies the cDNA using repeated cycles of denaturation, annealing, and extension.

• Denaturation: The cDNA is heated to separate the two strands of the double helix.

• Annealing: Primers bind to specific sequences on the cDNA strands.

• Extension: A DNA polymerase enzyme adds nucleotides to the primers, extending the cDNA strands.

The most well-studied and commonly used reverse transcriptase enzymes are derived from:

✓ HIV-1: Human immunodeficiency virus type 1.

✓ M-MLV: Moloney murine leukemia virus.

✓ AMV: Avian myeloblastosis virus.

✓ TERT: Telomerase reverse transcriptase.

DTI RT enzymes are primarily derived from M-MLV or AMV.

While both M-MLV and AMV reverse transcriptases are widely used, they have some distinct characteristics:

• RNase H activity: Both M-MLV and AMV possess RNase H activity, which is essential for removing RNA from the RNA-DNA hybrid during reverse transcription. However, M-MLV has a higher RNase H activity than AMV.

• Temperature sensitivity: M-MLV is generally more temperature-sensitive than AMV, meaning it may be less active at higher temperatures.

• Specificity: AMV is often considered more specific for certain RNA templates, while M-MLV may be more tolerant of secondary structures in RNA.

DNA Polymerase Activity: It synthesizes DNA strands using RNA as a template, primarily in the 5' to 3' direction.

RNase H Activity: This activity allows it to degrade the RNA strand that served as the template for DNA synthesis.

Thermostability: Reverse transcriptase can withstand high temperatures, essential for applications like PCR.

Processivity: Processivity refers to the ability of an enzyme to remain attached to its substrate (in this case, the RNA template) for multiple catalytic cycles without dissociating. A highly processive reverse transcriptase can synthesize long DNA strands efficiently.

Fidelity: Fidelity refers to the accuracy of the enzyme. Reverse transcriptase is generally accurate but can make errors.

Terminal Transferase Activity: Some reverse transcriptases also have terminal transferase activity, enabling them to add nucleotides to the 3' end of a DNA strand without needing a template. This activity is crucial for synthesizing specific DNA sequences, such as the long terminal repeats (LTRs) at the ends of retroviral genomes.