Key Difference – RT PCR vs QPCR
Polymerase Chain Reaction is a technique used to amplify a specific region of DNA in vitro. Due to the invention of this technique by Kary Mullis in 1983, scientists are able to make thousand to millions of copies of specific DNA fragments for research purposes. Currently it has become a common and routinely performed technique in clinical and research laboratories for a broad variety of applications. There are variations of traditional PCR technique such as RT PCR, nested PCR, multiplex PCR, Q PCR, RT – QPCR, etc. RT PCR and Q PCR are two important variations of PCR. The key difference between RT PCR and Q PCR is that RT PCR is used to detect gene expression through creation of complementary DNA (cDNA) transcripts from RNA while Q PCR is used to quantitatively measure the PCR products real time using fluorescent dyes.
CONTENTS
1. Overview and Key Difference
2. What is RT PCR
3. What is QPCR
4. Side by Side Comparison – RT PCR vs QPCR
5. Summary
What is RT PCR?
Reverse transcription polymerase chain reaction (RT PCR) is a variant of PCR which is used to detect RNA expression. It is a very important method for detecting mRNA expression in tissues. RT PCR is employed when the starting material of the sample is RNA. In RT PCR, the template mRNA is first converted into complementary DNA. This step is catalyzed by the enzyme reverse transcriptase and the process is known as reverse transcription. Secondly, traditional PCR is employed for the newly synthesized cDNA for the amplification.
RT PCR is a highly sensitive technique which requires a relatively small amount of RNA sample. RT PCR is commonly used in the diagnosis and quantification of RNA species, especially RNA viruses such as human immunodeficiency virus and hepatitis C virus.
Figure 01: RT PCR Technique
What is QPCR?
Quantitative PCR (QPCR) is a variant of PCR which is used to quantitatively measure the PCR products. It is also referred to as real-time polymerase chain reaction since it measures the amplification of real time using real time PCR machine. It is a suitable method for determining the amount of a target sequence or gene present in a sample. The interesting feature of QPCR is that it combines both amplification and true quantification into a single step. Therefore, the need for gel electrophoresis in detection can be eliminated by QPCR technique. QPCR uses fluorescent dyes to label PCR products during the PCR reactions which eventually lead to direct quantification. When the PCR products accumulate, the fluorescent signals are also accumulated and it will be measured by the real time machine. QPCR can be combined with RT PCR. It is known as RT – QPCR or QRT – PCR and is considered as a most powerful, sensitive and quantitative method for detection of RNA levels in the cells or tissues.
SYBR Green and Taqman are two methods employed to detect or watch amplification process of the real time PCR. SYBR Green method is carried out using a fluorescent dye called SYBR green and detects the amplification by binding the dye to produce double stranded DNA. Taqman is carried out using dual-labeled probes and detects the amplification by degradation of the probe by Taq polymerase and releases of the fluorophore as shown in figure 02. Both methods monitor the progress of the amplification process and report the quantity of the product real time.
Real time PCR has a wide variety of applications such as gene expression quantification, microRNA and noncoding RNA analysis, SNP genotyping, detection of copy number variants, detection of rare mutations, detection of genetically modified organisms, detection of infectious agents, etc.
Figure 02: Quantitative PCR technique
What is the difference between RT PCR and QPCR?
RT PCR vs QPCR |
|
| RT PCR is a technique used to detect gene expression by amplification. | QPCR is a technique which amplifies DNA and quantifies the PCR products in real time. |
| Involvement of Reverse Transcriptase Enzyme | |
| Enzyme reverse transcriptase is used for RT PCR. | Enzyme reverse transcriptase is not used for QPCR. |
| Use of Fluorescently Labeled Molecules | |
| Fluorescently labeled dyes or probes are not used for RT PCR. | Fluorescently labeled dyes or probes are used for QPCR. |
| Quantification of PCR product | |
| Unless coupled with QPCR, RT PCR does not quantify the PCR product. | QPCR quantitatively measure the PCR product. |
| Starting Material | |
| Starting material is mRNA. | Starting material is DNA. |
| Synthesis of cDNA | |
| Complementary DNA is produced during the RT PCR. | Complementary DNA is not produced during QPCR. |
Summary – RT PCR vs QPCR
RT PCR and QPCR are two versions of traditional PCR. RT PCR technique is performed for mRNA samples and it is driven by reverse transcription and production of cDNA. QPCR is used to quantify PCR products during the real time PCR thermal cycles using fluorescent dyes or labeled probes. In QPCR, the amount of PCR product is represented by the emitted fluorescent signals by the sample. RT PCR is popularly used as an amplification process while QPCR is commonly used as a quantification process. This is the difference between RT PCR and QPCR.
References:
1. Deepak, SA, KR Kottapalli, R. Rakwal, G. Oros, KS Rangappa, H. Iwahashi, Y. Masuo, and GK Agrawal. “Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes.” Current Genomics. Bentham Science Publishers Ltd., June 2007. Web. 03 Apr. 2017
2. Zeka, Fjoralba, Katrien Vanderheyden, Els De Smet, Claude A. Cuvelier, Pieter Mestdagh, and Jo Vandesompele. “Straightforward and sensitive RT-qPCR based gene expression analysis of FFPE samples.” Nature News. Nature Publishing Group, 22 Feb. 2016. Web. 03 Apr. 2017
3. Overbergh, L., A. Giulietti, D. Valckx, B. Decallonne, R. Bouillon, and C. Mathieu. “The Use of Real-Time Reverse Transcriptase PCR for the Quantification of Cytokine Gene Expression.” Journal of Biomolecular Techniques : JBT. The Association of Biomolecular Resource Facilities, Mar. 2003. Web. 03 Apr. 2017
Image Courtesy:
1. “Taqman” By User:Braindamaged – Own work by the original uploader (Public Domain) via Commons Wikimedia
2. “Reverse transcription polymerase chain reaction” By Jpark623 – Own work (CC BY-SA 3.0) via Commons Wikimedia