As many of you know, a good percentage of our customers are doing microbiome research.    Microbiomes, those localized communities of microorganisms that exist symbiotically with their immediate environment, can be found virtually anywhere; inside human colons, around plant roots, inside coral reefs and even within ant colonies.  These little microbial microcosms are a hot topic right now.   Recent studies have implicated their role for the wellbeing of people, animals, plants and entire oceans.   Variations in microbial numbers and diversity within animals have shown critical involvement in auto-immune disease, obesity, acne, tooth decay, pregnancy and even brain chemistry.   Plant studies have shown that symbiotic organisms impart resistance to drought, increase nutrient absorption and prevent attack by pathogens.

Here at MO BIO Laboratories, our contribution to this important research usually ends with the isolation of clean genomic DNA or RNA.  We know a lot about the beginning of the production line.  But what about what happens after the nucleic acids are extracted?  And how are they being used?   How is it that these conglomerations of microbial DNA/RNA can be converted into quantifiable information that is useful?   We thought we’d explore this further this week.

Amplicon and Whole Genome Sequencing

First and foremost, in order to understand how microbiomes might be important, we need to know what microbes are actually in there.   Not too long ago, the only reasonable way to do this was to culture the microbes and then try to identify the species by growing up a enough of them so that cell morphology, staining, antibodies, protein analysis or something similar could be used to identify the species.    There are two major problems with this method.    First, you lose all information about the relative concentrations of the microbes that were in there to begin with.   Secondly, many microbes will not grow under typical laboratory conditions and so you would miss their existence entirely.  With the invention of fast and cheaper DNA sequencing, the culturing step can be eliminated.   Microbial DNA and RNA can be directly extracted and isolated from a sample of soil, water, blood or stool, among other interesting bio-substances (our specialty).  Once the microbial nucleic acids are isolated they can be used to determine which species existed within the sample using sequencing.

There are two common sequencing methods currently being used for this purpose.  The first, referred to as targeted amplicon sequencing, requires knowing a little something about the community of microbes that might be in the sample to begin with.     Scientists make use of the gene for the 16S ribosomal RNA. (1)   It is the most highly conserved DNA in all cells but also contains a number of hypervariable regions that have diverged over time.   PCR is used to amplify those divergent sections of the microbial genomic DNA; and these differences make it possible to uniquely identify particular microbes.   By selecting primers that target conserved regions that flank the variable regions,  unique differences can be used to identify microbial species.    But while 16S ribosomal RNA is slightly different for virtually all species, no single hypervariable region can be used to distinguish all bacteria.  So scientists do the best they can by selecting those sets of primers that will characterize the most common sets of bacteria in their sample.    These are often referred to as “universal” primers.   But in reality there are no 100% universal primers.  This is one drawback of the method.

In the second method, whole genome sequencing, DNA or RNA is isolated directly from environmental samples and sequenced without using a PCR amplification step.   Sampled fragments of the whole genome or transcriptome are used to create libraries which are then sequenced in a pool.   The genomic sequence is reconstructed and these sequences are compared to known databases of microbial sequences, like MBGB or KEGG, using sequence comparative analysis programs like BLAST.  A disadvantage of this method is that not all microbial species have been sequenced or are accurately cataloged in these databanks.   So again, some microbes can be missed.

Analysis of Similarity

Once microbial populations are identified through nucleic acid isolation, sequencing and analysis, scientists then need to turn this into quantifiable information regarding what those population differences might mean.   To achieve this goal, studies make comparisons between the microbial populations of very well-defined groups.   Sometimes the groups are defined by their state of health, for example people with diabetes compared to those without.     They could also be defined by the locations that the samples were collected from, like soil from the Antarctic verses soil from the Russian Tundra.

Scientists compare the microbial population data from these well-defined groups and do what’s called an analysis of similarity (ANOSIM) to determine if the populations are more similar than they would be simply by chance.   This type of analysis can get quite tricky and in research it’s not just applied to microbial populations. But basically, one first generates data assuming there is no correlation between two data sets.   This random data is then compared to the actual population of data that exists.  From this analysis a statistical number called an R value is computed which indicates how similar or dissimilar two sets of data are when compared to what they would look like from pure chance.  When applied to microbial communities the R value, a number between -1 and +1 indicates how similar populations of microbes are between groups.   A number of +1 would indicate a very strong likelihood that microbial populations are similar in some way that would be very unlikely by chance.   A number of -1 would also be statistically significant but would imply that two populations of bacterial are dissimilar for some particular reason that is not by chance.  An R value of zero would indicate no relationship at all between the two populations.

Here is a simple example to illustrate.   Imagine drawing two large, identically sized circles in the sand, circle A and circle B.    You now have two different populations of bean bags: 20 red and 20 green.      Let’s say these represent two species of bacteria.   Now stand equally distant from the two circles and throw red and green bean bags into the circles in the sand.   You should end up with something close to 10 red and 10 green within each circle.   This is your random population, because there is nothing biasing the two populations either way.   Now do something different.   Use only your left hand to throw the green bean bags and your right hand to throw the red bean bags into the two circles.  Now compare the new population of bean bags in the two circles to the “random” one you did before.   If you see a difference, then you’ve discovered something.  Perhaps you don’t toss very well with your left hand.    If you don’t see any difference between the two sets of circles well then you’ve discovered something too.   There is nothing particularly significant about your throwing method. Scientists have a fancy name for this.   It’s called the “null hypothesis,” meaning there is no relationship between the two groups of measurements, that is right and left handed throwing have no affect.  If we were talking about two bacterial populations perhaps we’d be testing if a difference in temperature affected the similarity of two microbial groups or whether being in close proximity affected the similarity.

Okay, enough of all the technical stuff.  Next time we plan to discuss a recent and very cool publication out of Rob Knight’s lab which applies all of these techniques to learn about how family members’ microbiomes are affected by living with both their children and dogs. (Song et. al. Cohabiting family members share microbiota with one another and with their dogs, elifesciences.org, April 2013)   P.S.  It might make you think twice before you kiss your favorite pooch!

(1) Weisburg WG, Barns SM, Pelletier DA, Lane DJ (January 1991). “16S ribosomal DNA amplification for phylogenetic study”. J Bacteriol. 173 (2): 697–703.

We speak with many scientists who work with filtered water for isolating microbial DNA and RNA. Water samples can be difficult because of their typically low biomass (depending on the water source) and because these samples are often from precious and unique sources.

Why is molecular research on microbes in water difficult?

For some people, getting back to the original source of water may not be possible for months or even years. For example, we talk to scientists collecting samples at hydrothermal vents in the middle of the ocean, in the Antarctic, and in the Baltic Sea.  For some researchers, water samples may have been collected after a certain event, such as a flood or heavy rain and so the conditions of the water will not be the same in a week or even after a day. They need to get answers from every sample collected and they need it to accurately reflect the current microbial content.

Choosing a Filter:

People who want to determine the microbial communities of collected water will filter them onto filter membranes. The typical size is a 47 mm membrane. This is large enough to have a good flow but small enough to work for DNA or RNA extraction. If the membrane is too small (25 mm), it may clog if the water contains higher levels of debris and if it is too big (142 mm), it will need to be sliced up in order to fit in standard 5 ml and 15 ml tubes.  Ideally, the less handling and manipulations going on with the water filter, the more microbial DNA and RNA can be recovered.

To help make sure that the 47 mm filter membranes are extracted the most efficiently without needing to be sliced into small pieces, MO BIO Labs uses a 5 ml screw cap tube (see picture right). This tube allows for full access of the microbial side of the filter to be homogenized with the garnet grinding resin. We have found after thorough testing that this tube allows for maximal recovery of DNA from all types of filter membranes.

Another question we hear from customers is how to choose a type of membrane. There are many choices from polyethersulfone (PES) to mixed cellulose esther, MCE (cellulose acetate and cellulose nitrate) to polycarbonate to aluminum oxide. Each of these membrane types handle a bit differently and will give slightly different results after extraction.  It is important to remember that the different characteristics of a membrane also reflect its use for other applications such as direct culturing (PES, MCE) or light and electron microscopy (polycarbonate, aluminum oxide).  Overall selection of a membrane for DNA and RNA isolation is more dependent on pore size, sample volume, and retention of inhibitors such as pesticides.  In other words, more than one membrane type may work for your application.

In our experience here is what we found:

Polyethersulfone: Are one of the toughest membranes and can be handled more than the others. They dry quickly under vacuum making them easy to fold without tearing.   Both 0.45 and 0.22 micron pore sizes can be used but a 0.22 micron pore size is best when you want to filter large volumes of water with low microbial biomass because they can handle the longer harder pressure of the vacuum. For nucleic acid extraction, we can get yields equivalent to the mixed cellulose esther with the PowerWater® DNA and RNA Isolation Kits.

Mixed cellulose esther (cellulose acetate and cellulose nitrate): Are best for when a 0.45 micron pore size is needed.  We recommend the use 0.45 micron pore size if your water has a lot of debris and tends to clog or filter very slowly with 0.22 micron pore sized membrane. Cellulose membranes tend to retain water making them a little more difficult to handle.  The video below will demonstrate how we handle them in our lab.

There are several published studies demonstrating that pesticides and herbicides can bind to cellulose acetate and cellulose nitrate so if you are using water that may contain pesticides and herbicides, avoid using cellulose membranes.

Polycarbonate: This type of filter can be more difficult to work with due to its thinness and the ease at which it can wrinkle.  A 0.45 micron pore size is commonly used to prevent clogging.  Unlike the PES and MCE membranes, microbes in your water sample will sit on top of the membrane rather then inside.  This leads to clogging faster but also retention of smaller particles that would have been able to pass through.  We have found that for isolating DNA, less extreme bead beating will give you higher molecular weight DNA. If your sample is used for PCR only, then the stronger bead methods should be fine although expect a lot of shearing.

Aluminum Oxide: This type of filter is also known as an Anodisc™ filter membrane (Whatman).   It handles like a thin sheet of glass and will break up easily in any bead tube. Most labs are not using these due to the difficulty in transferring them to storage tubes. These are used with samples containing very low biomass such as ocean water.  They come in both 0.45 and 0.22 micron sizes.  Similar to the polycarbonate, microbes are retained more on top rather than within the filter, leading to easy extraction of DNA and RNA but also increased shearing with bead beating.

How to Handle a Filter Membrane:

Many of you out there probably already have a good technique for folding your filter membranes and placing them into a tube. For those of you who are new, or experiencing problems with this, here is a video we made in our lab to demonstrate the technique. This is a 0.45 micron mixed cellulose esther membrane.  Demonstrating is Heather Callahan, Ph.D, the scientist who created the PowerWater^® kits.

Summary:

We know how hard it is to work with different environmental water samples and how important it is to get every last drop of DNA or RNA. Our goal is to make sure you are successful at every step. When we developed the PowerWater and RapidWater products, we kept that in mind as we optimized each step; from the tube needed to grind in, to the matrix used for grinding, to the solutions used for removing inhibitors, to the binding chemistry, to the washing chemistry, and the final elution. If you have any questions on how to maximize your water filter DNA or RNA extraction, please send them to us and we’ll get back to you.  I can assure you, if you have a problem, we have the answer.

Ask the Water DNA and RNA Isolation Experts!

For more information on Water DNA and RNA Isolation products, go to:

PowerWater^® DNA Isolation Kits and RapidWater^® DNA Isolation Kits

PowerWater® RNA Isolation Kits

*This article is a re-post of the original which was published in October of 2009

Fun fact: Shakespeare's sonnets have been encoded in DNA

I would like to meet whoever is responsible for perpetuating the myth that scientists are a stuffy and dry bunch and introduce him or her to the amazingly creative and artistic scientists we have the pleasure of interacting with at MO BIO.  This month we invited our users to submit a poem about their MO BIO experience. The results were quite impressive. The topics ranged from our kit’s unparalleled ability to remove inhibitors to our great customer service. In return for their very kind words, we offered up a t-shirt and discount code.

Be sure to check out the video links of MO BIO employee’s reading the submitted poems.

**************** Read the rest of this entry »

Who hasn’t pondered the age old question: Which came first, the chicken or the egg?

Debates on this subject have kept philosophers busy for centuries, lending to rich discussions on everything from evolution of chickens to the beginning of life and the universe. One could ponder this question from a literal point of view and discuss the evolution of egg laying species and whether or not this pre-dates the appearance of chickens. Or one could set forth on a metaphysical journey and focus on the possibilities of how one life form can exist without its developmental precursor or how a precursor exist without its original maker?

Here at MO BIO, this discussion led us down a different path. The question we propose to you is: Which came first, the DNA or the protein? You need DNA to know what proteins to make and how to make them, but, you need proteins to synthesize more DNA, to transcribe the DNA into RNA, and to hold it all together.

Read the rest of this entry »

This week we want to discuss a technique that is very common but still causes many people to suffer from separation anxiety.  What could that possibly be? It is cleaning up dirty genomic DNA.

Here’s the scene: You have a precious soil sample collected from the roots of an ancient never-before seen orchid located on a remote island in the middle of the South Pacific. You isolated the DNA from microbes in this soil using a method other than the PowerSoil Kit. It’s still dirty and won’t amplify in PCR so it needs to be cleaned of humic acids and other humic substances. You need every last molecule for whole genome shotgun sequencing…. what do you do? Read the rest of this entry »

We’ve all got our groove on. Sampling and processing seems like a breeze despite it’s challenges.  The flow of efficiency has finally set in and it feels good! Read the rest of this entry »

We know many of our customers like to be selective about their RNA.  That’s because, most of our RNA technical questions involve a desire to retain or exclude certain varieties of RNA.   It’s not always possible to get what you want;  but sometimes by making slight adjustments to the extraction protocol, it is possible to get what you need.  In fact, in a previous MO BIO blog article [microRNA from Fresh Tissue and FFPE Samples using MO BIO Kits with Modified Protocols] we discussed how to bring in very small sized RNA when using our tissue extraction kits. Read the rest of this entry »

It’s Valentine’s Day and love is in the air at MO BIO! To celebrate this special (not so special for some) day, we thought we’d tell you about the cutest and nerdiest proposal of all time. Watch out guys, if your girlfriend sees this and is expecting a ring sometime soon, the pressure’s on!

You may have heard some buzz floating around recently about the guy who cleverly proposed to his girlfriend via agarose gel. Yes, we know, it’s incredible.  We wanted to ask him a few more questions about this amazing, geeky proposal,  so we tracked down the proposer, Eric (currently a Post-Doc at UCSD) , to get the whole story.

How did you two meet?

We were both graduate students at Stanford. We met at a grad student event and played volleyball together. We ran into each other again later at a department happy hour.

Where did you get the idea from? Read the rest of this entry »

Regional Climate Warming: West of the Antarctic Peninsula
Jan 17, 2013
68°00.2S X 69°30.8W
Sunrise  11:37pm
Sunset  9:49pm

MO BIO Labs is going to collaborate with our friends at the popular science blog site, Bitesize Bio to sponsor a couple of webinars for their readers.  Since MO BIO Labs has a primary focus in microbiology and ecology, we want to sponsor seminars that are going to highlight the research and scientists working in this exciting field.

As I was pondering potential speakers, I thougth about the women in science I’ve heard speak, who are an inspiration to me. But my experience is also limited being in the private sector. I don’t get to interact with as many women thought leaders as I would like. The question then became: who are the leading women in the field right now?  What women scientists do we have to draw insight and inspiration from? Read the rest of this entry »