Sample Prep Solutions for Microbiome Research

Editor's Notes

1. During cell lysis, which is the first step in DNA isolation, we use bead beating or mechanical lysis to break open microbes and release the DNA and this is the best way to lyse the most cells with the least amount of bias. In this process of bead beating, the inhibitors from the sample are released. In stool samples, this includes substances such as bile, bilirubin, and dead red blood cells as well as food as diet plays a major role in the content of the gut sample.
2. And this is where the Inhibitor Removal Technology comes in and is used to remove the inhibitory compounds before isolation of the nucleic acids so that the purification results in high quality DNA and RNA. This is a patented process that is utilized in all of the “Power” kits by MO BIO. To give you an idea of how important it is to remove these impurities, here is an example of how inhibitors will impact the perceived DNA yields and cause problems.
3. Here we are comparing the isolation of DNA from the same samples – prepared with and without IRT. The nanodrop data is what most people look at first and looking at this data, if we focus on yields only, when inhibitors are present it appears as if the samples prepared without inhbititor removal have higher DNA yields. If you look across at the purity readings, we see some problems- the combination of a low 260/230 ratio with a high 340 ratio tells us this co-contaminant is not just salt. It is compounds from the sample. If it were just salt, we’d see only a low 260/230 ratio. If we look at the plot data from the Nanodrop, we clearly see that the inhibitors are causing amplified absorbance across the entire range of wavelengths including the 260 reading. To confirm the nanodrop data we always run an agarose gel to check fro integrity and to make sure the yield data correlates with the gel picture. And clearly it does not. In fact the samples prepped with IRT have higher DNA yields than the samples without. We see some degraded RNA in these samples which will cause more 260 wavelength signal and a false DNA yield reading. Going into your next experiment, your input DNA is going to be inaccurate if you have DNA containing inhibitors. This will lead to inconsistences in the data going forward and problems with accuracy later when it comes time to analyze the results of qPCR or sequencing. So the take home message is that contamination removal is very important! This next slide demonstrates how inhibitors can impact qPCR.
4. https://www.whitehouse.gov/the-press-office/2015/03/27/fact-sheet-obama-administration-releases-national-action-plan-combat-ant https://www.beefusa.org/newsreleases1.aspx?NewsID=4966 Antibiotic resistance is a huge problem globally. This just gives you a background about some issues that we are experiencing just in the US with antibiotics resistance. Just in 2015, it was estimated by the CDC (Center for Disease Control in the US) that antibiotic-resistance would cause 2 million people to be sick, and about 23,000 deaths per year. In March of 2015, The Obama Administration released an action plan that spans over 5 years to help combat the continued emergence of antibiotic resistant bacteria both domestically and internationally. They set a lot of different goals with this plan, such as reducing unnecessary antibiotic use in patients and eliminating the use of certain antibiotics in meat animals that are just for growth promotion, and developing a more robust monitoring system for antibiotic usage. There are also more goals they plan to develop internationally as well and you can look through this entire plan online. On June 2, 2015, The National Cattlemen’s Beef Association participated in the White House Forum on Antibiotic Stewardship, and they were able to highlight how this industry is contributing to the fight against antibiotic resistance, and their plan to eliminate the use of medically-important antibiotics to increase feed efficiency or growth promotion**, which they plan to implement by December 2016. NOTES: **” intent of enhancing growth (to make animals grow faster) or to improve feed efficiency (the animals need less food to gain weight).”