Experts in qPCR
The standard for quantifiying target DNA sequences
Quantitative Real-Time PCR (qPCR) is the most widely spread method to measure gene expression levels, as well as detect and estimate the abundance of microorganisms in environmental and clinical samples.
qPCR is a highly specific, quick, and cost-effective method. It monitors the fluorescence within a PCR reaction and correlates it to the amount of target DNA synthesised in the sample. There are two different approaches:
- Absolute quantification assays: The initial concentration of the target DNA can be estimated by generating a standard curve of known concentrations.
- Relative quantification assays: The amount of target DNA in a test sample is compared to the amount of another reference DNA present in the same sample.
At AllGenetics we will advise you on the most suitable approach for your assay. We will help you with the experimental design, selection, and validation of target and reference DNA regions as well as the analysis of the resulting qPCR data.
How we work
Step 1: We isolate the desired nucleic acids (DNA or RNA) from your samples. We have adapted different DNA and RNA isolation protocols, depending on the starting biological material, as well as cDNA synthesis protocols.
Step 2: We perform qPCR to amplify and quantify target DNA sequences using in-house developed primer pairs and specific fluorescent probes or SYBR Green. A pilot qPCR experiment is carried out beforehand in order to check the specificity and efficiency of the assay to set up the qPCR conditions.
Step 3: We analyse the results of qPCR to estimate the abundance of target DNA in each of the samples analysed in relation to a standard curve (absolute quantification assays) or a panel of reference genes (relative quantification assays).
Please contact us for further information and pricing.
What you receive
A report with a summary of the methods followed.
The raw qPCR data, which will be delivered through our server.
The results of the analysis, including:
- A table with the estimated abundances or the expression levels of selected target sequences.
- A report of assay performance characteristics (melting curve analysis, PCR amplification efficiency, Cq values of non-template negative controls, etc).
If you require additional analyses, please let us know and we will do our best to meet your needs.
At no additional cost, our project specialists will be available to you after project delivery and during the editorial process of your scientific articles. We offer up to five 60-min sessions per project, depending on the project size.
What we need
Your samples appropriately preserved depending on the sample type (ethanol, silica gel, frozen, or another suitable preservation method). If required, we provide sampling kits and sampling collection guidelines to ensure that your samples arrive at our lab in optimal conditions.
We can start working by using either your tissue/environmental samples, or nucleic acid which has already been extracted.
If you are sending tissue samples, please make sure that they have been properly preserved for RNA isolation (liquid nitrogen, RNAlater®, or other suitable RNA preservation agents).
If you are sending RNA, please note that we require a minimum amount of 1 μg of high-quality total RNA for optimal results. Your RNA must be resuspended in nuclease-free water (concentration higher than 20 ng/μL per sample). RNA should be shipped in dry ice to prevent it from degrading.
If you are sending DNA, we require a minimum amount of 500 ng of high-quality DNA for optimal results. DNA must be at a concentration higher than 10 ng/μL per sample.
Please note that, depending on the specifics of your project, these requirements may vary. Please contact us for the specific requirements of your project.