Molecular biology method · BEGINNER LESSON
RT-PCR
RT-PCR, or reverse transcription PCR, is a method that first converts RNA into complementary DNA (cDNA) and then amplifies that DNA by PCR. It is used to study RNA levels or gene expression because the starting material is RNA, but the amplified product is DNA.
What RT-PCR is and what it measures
RT-PCR is a laboratory method that starts with RNA, usually messenger RNA (mRNA), and measures it indirectly by turning it into complementary DNA (cDNA) before amplification. PCR stands for polymerase chain reaction, a technique that makes many copies of a chosen DNA sequence. Because the first step changes RNA into DNA, RT-PCR is used to investigate RNA expression and to compare mRNA levels among samples.
[1] The polymerase chain reaction and pathology practice - PMC [2] A new reverse transcription-polymerase chain reaction method for accurate quantification [3] Polymerase Chain Reaction (PCR) - StatPearls - NCBI Bookshelf - NIH
How the method works
The first step is reverse transcription, which uses reverse transcriptase, an enzyme that copies RNA into cDNA. After that, PCR cycles repeat denaturation, annealing, and extension: the DNA strands separate, primers bind to matching sequences, and a thermostable DNA polymerase extends the new strands. Repeating these cycles produces many copies of the target sequence. RT-PCR can be done in separate steps or in one tube, and real-time RT-PCR can monitor product buildup during cycling with fluorescent signals.
[1] The polymerase chain reaction and pathology practice - PMC [3] Polymerase Chain Reaction (PCR) - StatPearls - NCBI Bookshelf - NIH [2] A new reverse transcription-polymerase chain reaction method for accurate quantification
Why results can differ between samples
RT-PCR accuracy depends on the quality of the RNA sample and on how efficiently reverse transcription and PCR amplification work. Small differences early in the process can become large after many cycles, which is why quantitative work must control reaction conditions carefully. Real-time PCR helps by tracking amplification during the reaction, but the method still depends on good primer design and efficient chemistry.
[1] The polymerase chain reaction and pathology practice - PMC [2] A new reverse transcription-polymerase chain reaction method for accurate quantification [3] Polymerase Chain Reaction (PCR) - StatPearls - NCBI Bookshelf - NIH
Limitations and sources of error
RT-PCR is sensitive, but that sensitivity also makes it vulnerable to contamination, which can create false-positive results. Poor design or poor optimization can give false negatives, and unintended amplification of related sequences can confuse interpretation. Crude or degraded nucleic acid preparations can reduce efficiency, and formalin-fixed, paraffin-embedded tissues often need short targets because the RNA or DNA is more broken down.
[1] The polymerase chain reaction and pathology practice - PMC [3] Polymerase Chain Reaction (PCR) - StatPearls - NCBI Bookshelf - NIH
SEE THE IDEA
How RT-PCR works
Starting material, often mRNA, that reflects gene expression
Reverse transcriptase copies RNA into cDNA
Denaturation, annealing, and extension copy the cDNA repeatedly
Product is measured after amplification or during real-time monitoring
The sample moves from RNA to cDNA, then through repeated DNA copying, and finally to detection of the amplified product.
RT-PCR measures RNA expression indirectly by converting RNA to DNA and then amplifying that DNA.
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Sources and further reading
This general lesson was generated from public source material. It explains a concept; it does not interpret a private paper or provide medical advice.