Quantitative PCR (qPCR), also known as Real-Time PCR, is a method that measures the number of copies of a DNA region defined by a particular PCR primer(s). Using this method, we specifically amplify the 16S amplicon and quantify the total bacterial content in each sample to determine total bacterial load.
This technique can also be used as a means to determine the feasibility of your microbiome study by providing an exact measure of bacterial abundance in your samples. We highly recommend using this technique prior to sequencing samples suspected to have low biomass, to determine whether these samples will result in positive amplification.
DNA extraction and quality control for qPCR
DNA is extracted from samples using the Qiagen MagAttract PowerSoil DNA KF Kit (Formerly MO BIO PowerSoil DNA Kit) optimized for the Thermofisher 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.