What is RT-PCR?

RT-PCR stands for Reverse Transcription Polymerase Chain Reaction. It's a laboratory technique used to detect and measure RNA.

The process involves two main steps:

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.

One-Step vs. Two-Step RT-PCR: Choosing the Right Method

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.

One-Step RT-PCR

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

• Advantages:
• Simplicity: Fewer handling steps reduce contamination risks and save time.
• Speed: Fast and convenient, especially for high-throughput or diagnostic applications.
• Consistency: All reagents are in one tube, minimizing variability between samples.

Two-Step RT-PCR

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.

• Advantages:
• Flexibility: Use the same cDNA for multiple targets, 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.

Which Should You Choose?

• Use one-step RT-PCR for a streamlined, rapid process (high-throughput diagnostics).
• Opt for two-step RT-PCR for complex or sensitive experiments requiring flexibility and multiple analyses from the same sample.

Principle

The principle of RT-PCR involves two main steps:

1. Reverse Transcription:
• RNA Conversion: RNA is converted into complementary DNA (cDNA) using reverse transcriptase.
• Primer Binding: A primer binds to a specific sequence on the RNA, providing a starting point for cDNA synthesis.
• cDNA Synthesis: Reverse transcriptase adds nucleotides to the growing cDNA strand, complementary to the RNA template.

Polymerase Chain Reaction (PCR)

• Exponential Amplification: Amplifies the cDNA using repeated cycles of denaturation, annealing, and extension.
• Denaturation: The cDNA is heated to separate the double helix.
• Annealing: Primers bind to specific sequences on the cDNA.
• Extension: DNA polymerase adds nucleotides to the primers, extending the cDNA strands.

Common Hurdles & How to Avoid Them

Common hurdles:

• Low cDNA yield
• Incomplete cDNA synthesis
• High background noise
• Inconsistent results
• Low sensitivity
• Inhibitors in the sample
• Non-specific priming
• Temperature sensitivity

How to avoid hurdles:

• Use high-quality RNA.
• Choose the right reverse transcriptase.
• Optimize reaction conditions.
• Check cDNA quality.
• Use a suitable RNA input amount.
• Purify RNA before use.
• Use appropriate primers.
• Consider using a kit.
• Store reagents correctly.
• Use a positive control.

Commonly Used Reverse Transcriptase Enzymes

Reverse transcriptase enzymes are commonly derived from:

• • HIV-1: Human immunodeficiency virus type 1.
• • M-MLV: Moloney murine leukemia virus.
• • AMV: Avian myeloblastosis virus.
• • TERT: Telomerase reverse transcriptase.

Key Differences Between M-MLV and AMV Reverse Transcriptase

• • RNase H activity: M-MLV has a higher RNase H activity than AMV, essential for removing RNA from the RNA-DNA hybrid.
• • Temperature sensitivity: M-MLV is more temperature-sensitive than AMV and may be less active at higher temperatures.
• • Specificity: AMV is often considered more specific for certain RNA templates, while M-MLV may tolerate RNA secondary structures better.

Key Features of Reverse Transcriptase

• • DNA Polymerase Activity: Synthesizes DNA strands using RNA as a template (5' to 3' direction).
• • RNase H Activity: Degrades the RNA strand from the RNA-DNA hybrid.
• • Thermostability: Can withstand high temperatures, which is essential for PCR applications.
• • Processivity: Highly processive enzymes can efficiently synthesize long DNA strands.
• • Fidelity: Generally accurate, though errors may occur.
• • Terminal Transferase Activity: Enables the addition of nucleotides to the 3' end of a DNA strand without a template.