Reverse transcription PCR (RT-PCR) is a technique combining reverse transcription of RNA into DNA (cDNA) with the amplification of specific DNA targets using polymerase chain reaction (PCR). This allows you to quantify RNA levels. Here's a breakdown of the process:
Steps in Reverse Transcription PCR (RT-PCR)
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Reverse Transcription (RT): Creating cDNA from RNA
- This is the crucial first step where RNA is converted into its complementary DNA (cDNA) using an enzyme called reverse transcriptase.
- Reverse transcriptase is an RNA-dependent DNA polymerase. It reads the RNA sequence and synthesizes a corresponding DNA strand.
- This step typically involves:
- Annealing: A primer, such as oligo-dT (which binds to the poly-A tail of mRNA), random hexamers, or a sequence-specific primer, binds to the RNA template.
- Extension: Reverse transcriptase adds DNA nucleotides to the primer, creating a cDNA strand complementary to the RNA. There are different types of reverse transcriptase enzymes, each with its optimal temperature and efficiency.
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Polymerase Chain Reaction (PCR): Amplifying the cDNA
- Once the cDNA is synthesized, it serves as the template for PCR amplification. PCR exponentially amplifies a specific DNA sequence.
- PCR involves repeated cycles of:
- Denaturation: Heating the reaction to separate the double-stranded cDNA into single strands.
- Annealing: Cooling the reaction to allow specific DNA primers to bind to the cDNA template. These primers flank the region you want to amplify.
- Extension: A DNA polymerase (e.g., Taq polymerase) extends the primers, synthesizing new DNA strands complementary to the template.
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Detection/Quantification (in some variations)
- Traditional RT-PCR (end-point PCR): The PCR product is analyzed after a set number of cycles, often using gel electrophoresis to visualize the amplified DNA. This method is less quantitative.
- Real-time or quantitative RT-PCR (qRT-PCR): This method allows for the monitoring of DNA amplification in real time. A fluorescent dye or probe is used to measure the amount of PCR product generated at each cycle. This provides quantitative data on the initial amount of RNA. Common dyes include SYBR Green, while probe-based methods involve using fluorescently labeled oligonucleotides that hybridize to the target sequence. qRT-PCR is often preferred for its accuracy and ability to quantify gene expression levels.
Types of RT-PCR
- One-step RT-PCR: Reverse transcription and PCR are performed sequentially in the same tube. This minimizes contamination and simplifies the process.
- Two-step RT-PCR: Reverse transcription and PCR are performed in separate tubes. This provides more flexibility for optimizing each reaction.
Applications of RT-PCR
- Gene expression analysis: Measuring the levels of specific mRNA transcripts to study gene activity.
- Diagnosis of infectious diseases: Detecting the presence of viral RNA (e.g., in COVID-19 testing).
- RNA sequencing library preparation: Generating cDNA for next-generation sequencing.
- Cloning: Creating cDNA copies of RNA for insertion into vectors.
Example
Imagine you want to measure the expression level of a specific gene in a cell sample. You would:
- Extract the RNA from the cells.
- Use reverse transcriptase to convert the RNA into cDNA.
- Use PCR to amplify the cDNA corresponding to your gene of interest.
- Use gel electrophoresis (for end-point PCR) or real-time PCR to quantify the amount of amplified cDNA, which reflects the original level of the gene's mRNA in the cells.
