Quantitative PCR (qPCR)
For information on sample collection, storage and transport please see our sample handling guidelines in the support section.
DNA extraction and quality control
DNA is extracted from samples using the MO BIO PowerSoil DNA Kit optimized for the KingFisher robot. This kit uses magnetic beads to specifically capture DNA while excluding organic inhibitors. Inhouse testing has shown this kit to have a good balance of DNA yield and quality as demonstrated on a variety of environmental sample types.
The MO BIO Powersoil DNA protocol is followed, with the addition of a bead beating step to facilitate lysis of particularly robust microorganisms. DNA quantification and quality checks are done via Qubit.
Quantitative PCR (qPCR, also known as Real-time PCR) is a method that measures the number of copies of a DNA region defined by PCR primers. This method is a great complement to microbiome sequencing, which targets the whole community, as it provides the absolute abundance of specific populations of interest. The specificity of the test is given by the sequences of the PCR primers which can target broad phylogenetic groups (e.g. phyla Firmicutes) or specific ones (e.g. Escherichia coli). qPCR can be used with phylogenetic markers (e.g. 16S rRNA, recA, etc) or functional genes (e.g. nifH).
After reviewing your particular study design our bioinformaticians can assist in designing or obtaining the correct primers for a potential qPCR assay.