Real-time polymerase chain reaction, also known as quantitative PCR (qPCR), is a widely used molecular biology technique that allows for the amplification and quantification of DNA/RNA in real-time. Real-time PCR is an improvement over traditional PCR because it provides quantitative data on the amplification process as it progresses. At REPROCELL we use Applied Biosystems ViiA 7™ real-time PCR platform. The following are the various services that REPROCELL can provide using real-time PCR:
- Quantification of DNA/RNA targets
- Single nucleotide polymorphisms (SNPs)
- Mutation detection
- Gene expression analysis
- Pathogen detection
- Copy number variations
- Validation of RNA sequencing data
- MicroRNA (miRNA) analysis
- Drug development research
- Food safety testing
The figure above shows amplification of human specific gene Rnase P for quantitation of DNA.
Quantification of DNA/RNA targets
Quantitation of DNA and RNA using real-time PCR is a widely used technique in molecular biology and genetics research to precisely measure the amount of a specific DNA/RNA sequence in a sample. The following are the steps involved:
- Verify availability of designed assays from Thermo Scientific
- Sample preparation, such as DNA/RNA from sample of interest
- Reverse transcription if using RNA
- Real-time PCR set up
- PCR amplification and fluorescence detection
- Quantification
The above figure shows a standard curve generated using known quantities of a DNA standard to quantitate DNA/RNA. We have used a human specific target gene (RNASE P) to quantitate DNA/RNA.
Detection of mutations/SNPs
Using real-time PCR or qPCR is a very sensitive and specific method and is particularly useful for detecting SNPs, point mutations, insertions, and deletions in a target DNA or RNA sequence. At REPROCELL the following steps are used to perform mutation/SNPs detection using qPCR:
- Verify availability of designed assays from Thermo Scientific
- Sample preparation, such as DNA/RNA from sample of interest
- Real-time PCR set up
- PCR amplification and fluorescence detection
- Data analysis
Designing specific primers and probes is very critical to detecting mutations and SNPs using qPCR. At REPROCELL we validate assays utilizing a polymorphism discovery panel from the Corielle Institute. We always include known positive controls for wild type and heterozygous samples in the experiments.
The figure above depicts an allelic discrimination plot where blue dots are homozygous CC, green dots are heterozygous AC and red dots are homozygous AA.
Gene expression analysis
Gene expression analysis is performed by the same, ViiA 7™ platform that is used for analyzing mutation detection and quantification of DNA/RNA. Unlike traditional PCR which starts with DNA, gene expression analysis starts with RNA. In this case, RNA undergoes reverse transcription PCR (RT-PCR) where it is reverse transcribed to complimentary DNA (cDNA) before PCR amplification. RT-PCR is a versatile and sensitive technique used in molecular biology, clinical diagnostics, and research. At REPROCELL we use RT-PCR to provide the following services:
- Gene expression analysis
- Quantification of RNA targets
- Validation of RNA sequencing data
- MicroRNA (miRNA) analysis
- Drug development research
- Pathogen detection
- Food safety testing
The figure above shows expression analysis of the PTGR1 gene in lung cancer samples and controls.
Pathogen Detection
RT-PCR is used in detecting pathogenic RNA viruses such as COVID-19, HIV, and influenza. RT-PCR has been extensively used in detecting COVID-19 during the recent pandemic. The viral RNA is reverse transcribed to cDNA and specific regions of the viral genome are amplified using RT-PCR, which enables the detection of the presence or absence of the virus. The same platform can also be used to detect DNA viruses, bacteria, and fungus using DNA.
The figure above shows detection of bacteria Helicobacter pylori in gastric cancer samples.
