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.
The reagents and components of the PureLink Microbiome DNA Purification Kit are produced according to high manufacturing standards, and they should not cause any issues with contamination. Keep in mind that many samples, including stool, saliva, and soil, contain substantial microbial communities that will produce very high yields of DNA. Therefore, there are likely to be many orders of magnitude less contaminating DNA than sample DNA. However, with some sample types like skin, the microbial community is small, and the amount of isolated DNA is low. This is when one needs to be cautious about potential contaminating DNA coming from the reagents or plastics utilized. Use only the best and most reliable kits, and be very careful with components of the entire workflow: pipets, tips, water, and downstream assays such as qPCR. Everything should be extremely clean, and ideally all manipulations should be done in a clean hood by highly skilled scientists.
This PureLink kit was developed using a wide range of sample types: primarily stool, but also soil, urine, saliva, swabs, and other samples. In addition to analysis of bacterial communities, other components of the microbiome community were explored as well, including fungi. The kit contains specialized beads that efficiently break open cell walls and membranes with the help of heat and chemical lysis. This triple lysis approach works well for species with thick cell walls that are difficult to lyse, such as fungi. And any stool-derived inhibitors are rapidly eliminated with the included novel clean-up buffer, resulting in DNA that is compatible with any type of downstream analysis.
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.
Yes, the PureLink kit was successfully used for isolation of microbial DNA from human, mouse, rat, horse, cow, dog, cat, and other types of stool. Despite the fact that these sample types are very different in consistency, microbial composition, and presence of food debris and inhibitors, the kit enables fast and efficient isolation of substantial amounts of clean microbial DNA.
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.