The main difference between real-time and reverse transcriptase PCR is that real-time PCR gives faster and more detailed results and helps to quantify nucleic acids, whereas RT-PCR helps to detect and amplify cDNA.
PCR, or the polymerase chain reaction, is a laboratory technique to produce millions and billions of copies of a specific segment of DNA rapidly to study it in detail. Short synthetic DNA fragments called primers are used to select a part of the genome which is to be amplified, and multiple rounds of DNA synthesis are performed in order to amplify the selected part. Moreover, there are three steps in the polymerization chain reactions: denaturation, annealing, and extension. PCR also has many applications, including the identification of infectious agents and the analysis of ancient samples of DNA. There are different types of PCR. Real-time PCR and reverse transcriptase PCR are two such types.
Key Areas Covered
1. What is Real-Time PCR
– Definition, Features, Results
2. What is Reverse Transcriptase PCR
– Definition, Features, Results
3. Difference Between Real-Time and Reverse Transcriptase PCR
– Comparison of Key Differences
Real-Time PCR, Reverse Transcriptase PCR, qPCR, RT-PCR
What is Real-Time PCR
Real-time PCR measures the accumulation of amplification product as the reaction progresses by quantifying the product after each cycle in real-time. Real-time PCR is often known as qPCR or quantitative PCR and can be used quantitatively and semi-quantitatively. Moreover, there are two predominant methods for the detection of products of PCR. They are non-specific fluorescent dyes (which intercalate with any double-strand DNA) and sequence-specific DNA probes, which consist of oligonucleotides. Additionally, these oligonucleotides are labeled with a fluorescent reporter, which permits detection only after the hybridization of the probe with its complementary sequence.
In this type of PCR, data is recorded during the amplification process at the exponential phase. Moreover, this is a high-resolution technique that takes less time to deliver the result. During qPCR, different peaks related to different DNA fragments are seen. To analyze gene expression, one must identify and detect pathogens, identify and quantify mutations and quantify the DNA present in the sample. Furthermore, real-time PCR gives a more detailed and faster result.
What is Reverse Transcriptate PCR
Reverse transcriptase (RT-PCR) helps to detect and amplify cDNA. The reverse transcription has two steps. The reverse transcription process is the first step, while the second step is the amplification of the desired DNA sequence by PCR. In reverse transcriptase, the steps followed are the same as the steps in PRC except for the additional step, reverse transcription. Here, using reverse transcriptase, complementary DNA (cDNA) is produced first. This cDNA also acts as a template for the standard amplification process by PCR.
Furthermore, to detect RNA, scientists use reverse transcriptase, and measure the amount of RNA using the technique RT-qPCR. Also, scientists utilize this combined RT-PCR and RT-qPCR technique in the analysis of gene expression and viral DNA.
Difference Between Real-Time and Reverse Transcriptase PCR
Real-time PCR is a technique to measure the amount of DNA or RNA present in a sample by amplifying specific DNA or RNA sequences in real-time during the PCR reaction and measuring the amount of amplification product after each cycle using fluorescent probes. On the other hand, reverse transcriptase PCR is a technique to detect RNA molecules in a sample by converting them into complementary DNA (cDNA) using the enzyme reverse transcriptase.
Real-time PCR helps to quantify the nucleic acids, whereas RT-PCR helps to detect and amplify cDNA.
Furthermore, real-time PCR gives faster results than the reverse transcriptase PCR
Details of the Result
Real-time PCR gives a more detailed result than reverse transcriptase PCR.
In brief, PCR is a technique that helps to produce millions and billions of copies of DNA rapidly. There are different types of PCR techniques, and real-time PCR and reverse transcriptase PCR are two of them. The main difference between real-time PCR and reverse transcriptase PCR is that real-time PCR helps to quantify the nucleic acids, whereas RT-PCR helps to detect and amplify cDNA.
1. “Reverse transcription polymerase chain reaction” By Poshul – Own work (CC BY-SA 4.0) via Commons Wikimedia
2. “Workflow of a process involving real-time PCR” By Tzachi Bar – Own work (CC BY-SA 4.0) via Commons Wikimedia