Complete Q&A from microbiome research webinar

Your colleagues had interesting questions during the webinar “Transform your microbiome research, new isolation and analysis methods”—so we’ve collected and answered them here. If you have any other questions, please feel free to visit our support centers.

Yes, the PureLink Microbiome DNA Purification Kit will work for your research needs. There are 7 protocols available online, one specifically developed for swabs. The kit does not include swabs. Once you choose the swab protocol or transport media protocol (depending on which type of swabs you use—some are for shipping in dry form and some come with a medium to keep the swabs from drying out in transit), you will find some suggested swab brands/types that we have tested with this kit.

For highly pure samples, both fluorometers (such as Qubit™ Fluorometers) and spectrophotometers (such as NanoDrop™ Spectrophotometers) are successfully utilized and give very similar readings. However, when dealing with DNA or RNA samples that have high levels of contaminants (e.g., salts, proteins), Qubit fluorometers typically provide more accurate readings because they use dyes that are very specific to the analyte of interest. We have seen some challenging microbiome and FFPE samples where NanoDrop instrument readings were up to 2 times higher than those for Qubit instrument readings, because the NanoDrop instrument overestimated DNA concentration. Increasingly, laboratories use both instruments, as they provide complementary information: Qubit instruments are more accurate and specific for determining nucleic acid concentration, while Nanodrop instruments provide valuable information regarding purity, e.g., A260/A280, A260/A230, and the entire spectrum.

The bulk of DNA extracted from human stool is of microbial origin. We estimate that, depending on the sample, at most 20% of the total DNA isolated is host DNA while the remaining >80% is microbial DNA. For most microbiome projects, the DNA derived from bacteria, fungi, etc., is of primary interest; but for some projects—such as exploration of colon cancer diagnostics—analysis of human DNA derived from stool is the focus.

We simplified the protocol for scientists who are only interested in isolating human DNA from stool, because mammalian cells are easy to lyse and DNA isolation can be performed faster. Microbes have thick cell walls and are, in general, substantially more difficult to lyse. Thus, additional steps are required to ensure efficient disruption of all of the microbial species.

There are several options. One option is to dilute your DNA sample 10- or 100-fold prior to PCR; this will dilute out the contaminants, but the PCR signal in many cases will still be strong. Another option is to use “tough” enzymes that were specifically designed and optimized to be effective in harsh environments and to work in the presence of high levels of inhibitors (e.g., TaqPath™ products). And finally, you can use a BSA additive for the PCR reaction. In most cases, if used at an appropriate concentration, BSA can rescue your sample.

We analyzed 3 liquid versions: Amies™ Transport Medium, Stuart™ Transport Medium, and Cary Blair™ Transport Medium. These are the most popular types of media, utilized by scientists and medical specialists worldwide for many years.

When stool, body fluids, swabs, and other samples cannot be processed fresh, freezing the samples at –20°C or –80°C is the best way to preserve them. Try to minimize freeze-thaw cycles. For many samples, storage at 4°C for up to one week is also fine. One should be careful with commercially available DNA-stabilizing reagents, as some of them contribute very little to sample stability and actually can negatively impact the downstream nucleic acid isolation or make the workflow more challenging or lengthy.

For Research Use Only. Not for use in diagnostic procedures.