Key Differences Between PCR, RT-PCR and qPCR

Differences Between PCR, RT-PCR and qPCR Targeted Amplification Techniques

RT-PCR and PCR refer to the same technique and coronavirus detection test. The full name is Reverse Transcription Polymerase Chain Reaction test but is often abbreviated as PCR test only.

Is RT-PCR the same as PCR test?

Yes, RT-PCR and PCR refer to the same test. The full form of RT-PCR is Reverse Transcription Polymerase Chain Reaction. It is often simply abbreviated as PCR (Polymerase Chain Reaction) test.

To explain briefly, a PCR test detects genetic material from viruses, bacteria or other microorganisms using a technique termed as amplification. The RNA genetic material is first converted into DNA through a process called reverse transcription (RT), hence also named RT-PCR. The key advantages are that even tiny quantities of pathogen DNA/RNA can be rapidly amplified into measurable quantities.

RT-PCR and PCR both indicate the same common coronavirus detection test that works by detecting the virus's genetic code. This is the most accurate COVID-19 confirmatory test. The methodology amplifies viral genetic material targeting unique virus RNA sequences if present in the patient sample.

What is the Difference Between PCR and qPCR?

The key difference between PCR (polymerase chain reaction) and qPCR (quantitative PCR) is:

  • Detects presence of target DNA sequence
  • Qualitative test (presence or absence of DNA)
  • Does not allow quantitation of DNA
  • Quantifies DNA concentration
  • Quantitative test
  • Measures exact DNA levels or gene expression
  • More sensitive and precise quantification

In PCR, the DNA sequence gets exponentially amplified until reagents are exhausted giving a qualitative positive or negative result for sequence detection.

In qPCR, fluorescent tags allow quantification of DNA during each replication cycle. Result is exact concentration rather than just qualitative positivity of sequence presence.

While both PCR and qPCR amplify target DNA, qPCR technique additionally monitors amplification progress in real time. This allows precise target DNA quantification useful for gene expression analysis, microorganism load detection etc needing exact measured levels.

In summary, both multiply DNA but qPCR also enables simultaneous sensitive quantification useful for certain applications requiring numerical results.

Is Real-Time PCR and Reverse Transcriptase PCR Same?

No, real-time PCR and reverse transcriptase PCR are not the same. The key differences are:

Real-time PCR:

  1. Amplifies and quantifies DNA sequences
  2. DNA is the starting genetic material

Reverse transcriptase PCR (RT-PCR):

  1. Amplifies RNA genetic material after converting to cDNA
  2. RNA is the starting genetic material

Real-time PCR is used to directly detect and quantify DNA sequences like from bacteria, viruses etc. It uses fluorescent dyes to measure DNA amplification at each cycle in real-time.

RT-PCR first converts RNA into complementary DNA (cDNA) using reverse transcriptase enzyme. The cDNA is then amplified like normal PCR. It is used to study gene expression by amplifying RNA.

While both techniques allow amplification and quantification, real-time PCR works with DNA and RT-PCR converts RNA into cDNA first before amplification.

So in summary, the central difference is real-time PCR amplifies DNA while RT-PCR amplifies RNA sequences after reverse transcription into cDNA. They have different applications based on studying DNA or RNA.

What are the Steps of RT-PCR?

The main steps involved in Reverse Transcription Polymerase Chain Reaction (RT-PCR) are:

  1. RNA extraction: First, RNA is extracted and purified from the sample (cells, tissues etc). Good quality RNA is vital.
  2. Reverse transcription: The extracted RNA is then converted into complementary DNA (cDNA) by an enzyme called reverse transcriptase.
  3. Initial denaturation: The cDNA sample is heated to separate the double-stranded DNA into single strands.
  4. PCR amplification with primers: Repeated thermal cycling helps amplify target cDNA exponentially using sequence specific primers.
  5. Final extension: The final elongated strands are completed by supplying reagents and optimal temperature.
  6. Visualization: The amplified PCR products are finally visualized through agarose gel electrophoresis for confirmation before quantitation and analysis.

So in summary, the key stages are:

  1. RNA extraction
  2. Conversion of RNA into cDNA
  3. PCR amplification of target cDNA
  4. Visual detection and quantitation

This allows even tiny quantities of RNA to be studied by first amplifying it as cDNA using RT-PCR technology.

What are the Common Tests Done on Each PCR, RT-PCR and qPCR?

Here are some of the common tests done using PCR, RT-PCR and qPCR techniques:

PCR (Polymerase Chain Reaction)
  • Pathogen detection (bacteria, viruses)
  • Genetic disease testing
  • DNA fingerprinting/forensics
  • Genetic compatibility testing
  • Detecting DNA modifications
RT-PCR (Reverse Transcription PCR)
  • Gene expression profiling
  • Viral load detection
  • Cancer biomarker analysis
  • MicroRNA analysis
  • Diagnosing viral infections
  • Validation of RNA interference
qPCR (Quantitative PCR)
  • Viral load quantification
  • GMO detection/quantification
  • Gene expression level analysis
  • Microbial detection/quantification
  • Copy number variation analysis
  • cDNA quantification
  • Biomarker validation

In summary, while the underlying technology of amplification is similar, PCR, RT-PCR and qPCR are applied in different types of qualitative detection versus precise quantitative analysis assays based on the need.

The high sensitivity allows minute traces of DNA/RNA to be multiplied exponentially for improved precision and accuracy in multiple domains - from diagnosis to research.

From pathogen detection to precision medicine, polymerase chain reaction (PCR) based genomic amplification technologies provide unmatched sensitivity in identifying DNA/RNA signatures across diverse applications - right from diagnostic testing to cutting-edge research domains. Though the fundamentals of exponential amplification remain unchanged, continuous innovations focussed on accuracy, speed and affordability establish PCR/RT-PCR/qPCR as integral pillars of both healthcare and life science workflows now and in the foreseeable future.
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