Quantitative PCR is a modification of the conventional PCR technique that allows quantification of the initial amount of a target fragment present in the original sample, either DNA or RNA (cDNA).
Two quantitative PCR methodologies can be applied in the Genomics Section: real-time PCR and digital PCR.
qPCR in Real Time (qRT-PCR)
Theoretically, there is a quantitative relationship between the amount of the initial target sequence and the amount of PCR product in any amplification cycle. By including fluorochromes, the real-time progress of the amplification reaction can be monitored by measuring the fluorescence released each time a copy of the template DNA is made, since the increase in fluorescence intensity is proportional to the amount of DNA generated.
Applications:
- Gene expression studies: absolute quantification with standard curve and relative quantification with endogenous control gene.
- SNP detection.
- Detection of food pathogens.
- Determination of viral load.
- Determination of minimal residual disease.
- Allelic discrimination studies, etc.
Equipment
The equipment available in the laboratory is an Applied Biosystems StepOne Plus model that allows the analysis of 96 samples. Simultaneous detection of more than one fluorophore can be performed and, therefore, the detection of more than one amplicon in the same tube. Assays can be performed with SYBR Green or with labelled probes.
Primer Express software is also available for the design of probes and primers.
Services offered
The section offers the following services to researchers:
- Advice on experiment design and data analysis.
- Design of probes and primers with Applied Biosystems' Primer Express software.
Sample preparation and submission
The user is responsible for carrying out the PCR reactions. The sequencing laboratory will provide the necessary tubes or plates to be used in the equipment, as well as the SYBR Green PCR master mix or TaqMan PCR master mix reagents, when requested.
Users will be required to complete an initial training session in the SCSIE laboratory before being able to use the equipment.
Delivery of results
Each researcher will be given a copy of the data file, which can be analysed by installing the software provided by the technicians of the Genomics Section.
Digital PCR (dPCR)
This technique allows the absolute quantification of a DNA or cDNA without the need to use a standard curve or an endogenous control gene. The DNA or cDNA template molecules in the sample are individually separated into partitions that function as microchambers in which PCR reactions are performed in parallel. Specific products are detected using the same fluorochromes as in real-time qPCR. When the target sequence is present in a partition, it is amplified and a signal is generated (positive partition). If amplification does not occur, it is considered a negative partition. Applying a Poisson distribution, the ratio of positive and negative partitions is used to determine the absolute number of target molecules in the sample.
Applications:
- Absolute quantification of viral load
- Quantification of pathogens
- Generation of reference standards
- Quantification of gene expression
- Detection and quantification of GMOs
- Detection of low frequency mutations
- Quantification of donor/recipient chimeras
- CNV analysis
- Confirmation of variants detected by NGS
Equipment
The dPCR equipment available in the Genomics Section is the QuantStudio 3D model from Life Technologies. The system for obtaining the partitions and performing the amplifications is a 20,000-well microfluidic chip.
The system allows the detection of more than one fluorophore simultaneously and can use SYBR Green or labelled probes.
Services offered
- Advice on the design of the experiment and data analysis.
- Performance of the experiment.
Sample preparation and delivery
- Prior consultation with the section staff.
