We receive many calls from scientists isolating DNA from fecal samples, so I thought I’d share some tips on what we’re currently recommending for stool.

Stool is a rich microbial habitat that has a unique community to each individual.   The variation in stool samples is caused by many factors. Diet can play a major role in composition and texture. Stool taken from a person with a vegetarian diet may have a higher level of polysaccharides and fiber from plant material, and therefore more inhibitors. Also, the use of antibiotics will disrupt the bacteria living in the gut and cause changes in the sample content and consistency. And of course, some foods, especially those eaten raw, for example, sushi, can impact the gut microbiome.

Just like soil, stool samples are packed with microbes that can vary depending on where the sample was collected,  are rich in inhibitors, and can have different textures which will impact extraction. And most likely, if you are working with top soil, there will be some fecal material in your sample generated by earth worms, bird droppings, and other small animals.  This is the reason the MO BIO kits for soil are perfectly adapted for stool.

MO BIO has several options for working with fecal material depending on the level of complexity of the sample type. The UltraClean Fecal DNA Kit is good for samples that contain a low level of inhibitors. For maximal removal of PCR inhibitors, the PowerSoil Kit is always our first choice.  It is also the method used for processing samples in the Human Microbiome Project but with some changes to the protocol.

Protocol:

The SOPs for the Human Microbiome Project are listed on this webpage.   The nucleic acid purification protocols are at the very bottom under the section “Metagenomic WGS”.

Changes to the PowerSoil DNA Isolation Kit are described on page 7-24 of the Manual version 11 (page 65 of the PDF).

For this protocol change, the HMP researchers are collecting 2 ml of specimen into a 50 ml Falcon tube and adding 5 ml of Bead Solution (cat# 12988-10-BS) and then distributing the sample into several samples.

We recommend, for people who want to process just a single sample, to put the stool sample directly into the PowerBead tube that comes in a PowerSoil Kit and vortex for 30-40 seconds to disperse the samples.

Next, a heating step is added in to enhance microorganism lysis:

Heat at 65ºC for 10 minutes, then at 95ºC for 10 minutes. Samples may be stored frozen at -80ºC prior to DNA extraction. 

Following this treatment, the standard protocol for the PowerSoil DNA Isolation Kit is followed with a slight change at step 12 to centrifuge after Solution C3 for 2 minutes instead of one minute.

DNA is eluted in 100 ul and used for next generation sequencing.

For stool samples with the consistency closer to clay, another option that may work better is the PowerLyzer PowerSoil DNA Isolation Kit.  We will be publishing research shortly that demonstrates the improved yields of DNA from clay soil using glass bead tubes. I’ll come back and post the link when it is ready.

How it Works:

One theory as to why the PowerSoil and UltraClean Fecal Kits work so well for microbial DNA isolation from fecal samples, (besides the IRT steps for removing inhibitors), may be that the final eluted DNA is enriched for microbial DNA, making PCR more sensitive. The heating steps used above will lyse the human cells first, releasing the free DNA into the sample during the bead beating steps. Microbes need to be bead beaten to break open. The free human DNA will be sheared significantly during bead beating while the microbial DNA stays high molecular weight.

For binding DNA in these kits, the binding solution (C4) captures the high molecular weight DNA only and releases RNA and small broken DNA. The broken pieces of human DNA should wash out, leaving a sample that contains a higher proportion of microbial DNA to human DNA. This means more microbial DNA goes into every reaction. For next generation sequencing, you’ll get more information from each sample from the microbiome only.

What about RNA?

For RNA from stool, the RNA PowerSoil Total RNA Isolation Kit has been referenced (see below). This method allows for a higher starting volume so that more total RNA may be recovered from each prep. If you are looking for a method to isolate RNA from small amounts of stool sample, contact us (technical@mobio.com) and we can discuss the options and provide a free sample for evaluation.

Human Microbiome  references:

Dominguez-Bello, M.G., E.K. Costello, M. Contreras, M. Magris, G. Hidalgo, N. Fierer, R. Knight. In Press. Delivery mode shapes the acquisition and structure of the founder microbiota across multiple body habitats in newborns. Proc. Natl. Acad. Sci. USA. Jun 2010; 10.1073/pnas.1002601107.

Fierer, N., C.L. Lauber, N. Zhou, D. McDonald, E.K. Costello, R. Knight. 2010. Forensic identification using skin bacterial communities. Proc. Natl. Acad. Sci. USA. 107: 6477-6481.

Costello, E.K., C.L. Lauber, M. Hamady, N. Fierer, J.I. Gordon, R. Knight. 2009. Bacterial variation in human body habitats across space and time. Science. 326: 1694-1697

RNA PowerSoil Kit and stool

Exfoliated Cells in Stool: A Source for Reverse Transcription-PCR–Based Analysis of Biomarkers of Gastrointestinal Cancer
Ying Jie Yu, Adhip P.N. Majumdar, Jordan M. Nechvatal, Jeffrey L. Ram, Marc D. Basson, Lance K. Heilbrun, and Ikuko Kato. Cancer Epidemiol. Biomarkers Prev.,Feb 2008; 17: 455 – 458.

One problem with next generation sequencing projects is the handling of massive amounts of sequencing data that must be organized, cleaned up, assembled, and analyzed. Sequencing read lengths using the 454/Roche instrument are between 100-400 bp in length and the sequencing of an entire genome can generate millions of pieces of sequence that must be assembled. 

For example, researchers at Bielefeld University in Germany used a single sequencing run on the Genome Sequencer FLX system to completely assemble and characterize the genome of Corynebacterium kroppenstedtii.  In 7.5 hrs, they generated over 500,000 shotgun reads with greater than 100 million bases that were assembled into a contiguous genomic sequence with a total size of 2,446,804 bp. Can you imagine the bioinformatics required to assemble that much information in one day?  It is a primary concern for next generation sequencing labs using this innovative technology for microbial community analysis in rare environmental samples. Easy to use computing programs are desperately needed to make data interpretation manageable and fast. 

The purpose of the PANGEA (Pipeline for analysis of next generation amplicons) program is exactly this. This month in the July issue of The ISME Journal (4, 852-861, July 2010), Adriana Giongo from the lab of Eric Triplett at the University of Florida in Gainesville published a study demonstrating the functionality of a new set of computing tools for making analysis of next generation sequencing data faster and easier. PANGEA is written in Perl and can be run on Mac OSX, Windows, or Linux.

What is PANGEA and what is it for?

PANGEA is used to compile the huge datasets generated after 454/Roche next generation amplicon sequencing. In this publication, PANGEA was used to demonstrate assembly and annotation of 16S rRNA libraries for the purpose of microbial identification in a metagenomic analysis. To make next generation sequencing more cost effective and higher throughput, barcoding techniques are used to tag the 5′ end of DNA samples so that multiple libraries may be run simultaneously and then later organized and assembled. PANGEA takes the sequencing data directly from the 454 and performs all the necessary steps to generate files used for sequence identification by BLAST. The program includes statistical analysis to look at diversity of communities as well.

Although this study is a microbial analysis, PANGEA may be used to perform the bioinformatics for any barcoded amplicon sequencing project and can identify the origin of the sequences if an appropriate database for the gene of interest is available.

The source codes are freely available at pangea-16s.sourceforge.net and Microgator.org.

Samples:
To demonstrate the utility of PANGEA, the authors performed two different studies. The first was an analysis of fecal DNA from rats and the second was a study of surface soils collected at Hawaii Volcanoes National Park in May 2008.

Sequencing:
Sequences were generated using the GS FLX 454 DNA pyrosequencer (454 Life Sciences, CT). A total of 89,847 reads were obtained from the rat fecal samples and 275,529 reads were obtained for the soil samples. Sequences were trimmed to remove short reads or low quality scores using a script called Trim2 (Huang et al., 2003).

A perl script called barcode.pl was used to separate the reads into their respective groups and then remove the barcode from the read and insert a number at the beginning of each sequence.

Megablast is part of the BLAST package at NCBI and was used to analyze the final trimmed and barcode separated sequence. These sequences were then phylogenetically classified using TaxCollector to attach taxonomic information of the closest bacterial relative to each sequence, according to the best match in a modified bacterial RDP-II database.

Statistical Analysis:

The numbers of sequences were normalized using selector.pl and the Shannon diversity index was determined using a script called shannon.pl.

Results

High throughput sequencing of two 16S rRNA gene datasets was performed to demonstrate the utility of PANGEA for rapid characterization of microbial communities. The rat dataset which contained ~89,000 sequences was analyzed in only 24 hrs using a Mac Book Pro with 2.4 GHz Intel Core 2 Duo and 4GB 667 MHz DDR2 SDRAM. 

One of the benefits of PANGEA over other programs, such as CD-HIT, is that PANGEA identifies sequences before clustering instead of after. This is important because it allows every sequence to be classified at the genus and species level first whereas clustering before identification results in loss of representation of smaller sequence reads. If sequences are clustered first, the clusters are created according to sequence length and some sequences may be placed into categories that are not a good fit. In this experiment with rat fecal samples, fewer and sometimes different genera were observed using the CD-HIT method vs. the PANGEA method of data organization.

Another major benefit of PANGEA is that it normalizes the data before community analysis so that the number of sequences in each sample within a barcoded set is identical. This minimizes the effects of variation between the number of sequencing reads for each barcoded data set. Variation in number of reads may be due to errors in quantification of the genomic DNA at the start or problems with samples lacking a barcode during the library construction.   Normalization of the data sets is crucial for accurate diversity measurements using the Shannon method.

The authors conclude their paper with a thorough analysis of the differences between PANGEA and another data analysis tool called the RDP Pipeline. A few of the major advantages of PANGEA mentioned include that PANGEA is a stand alone tool not dependent on a web interface.  Uploading very large datasets to a web based program is not only slow, but also leads to concern about confidentiality. In addition, the user does not need to wait in line for their project to be finished because they have complete control of the analysis and the databases used. Another advantage of PANGEA over the RDP Pipeline is that PANGEA is fully automated and does not require the user to keep feeding the program files after each step.

More information on the PANGEA workflow and command lines for the program are described in the paper available online.

Summary:

With the explosion of the use of next generation sequencing in metagenomic projects come the need for bioinformatic tools that can organize huge datasets accurately. When files containing data for 300,000,000 sequences are generated in a single run, it is easy to make mistakes and lose critical information if the tools for analysis are not efficient. After reading this report by Adriana, it is clear that PANGEA is an easy to use program for the handling of barcoded amplicon next generation sequencing datasets.

As the U.S. struggles with the after effects of the oil spill on tourism, animal health, and food safety due to the largest environmental disaster in U.S. history, microbiologists are stuggling to determine the scope of damage to microbial diversity in the ocean water and sediment.

The explosion of the Deepwater Horizon oil rig on April 20th, 2010 that resulted in the deaths of 11 workers is still not capped and leaks an estimated 1.47 million gallons of oil a day (see ticker below).  Current estimates of total oil spilled as of July 1st are at a record 140.6 million gallons, and with weather conditions this time of year, reports of oil and tar balls washing up along all the Gulf states (Alabama, Florida, Louisiana, Mississippi, and Texas) are being confirmed.

Ecological Impact

In terms of the ecology and effect on wildlife in the Gulf, as of July 5th, the total counts of dead birds, sea turtles, and mammals, as reported in the Fish and Wildlife collection report, are at 1387, 444, and 53, respectively, and these numbers do not include the animals that were collected alive and either cleaned and released, or those that may have died later. 

What will take a lot longer to assess is the ecological impact of the oil spill on the microbial life in the ocean and sediment. The formation and existence of microbial mats and biofilms on the ocean floor, hydrothermal vents, or living symbiotically on the surface of sea life will be dramatically changed or impaired.  In addition, the rare biosphere of marine microbes that makes up the vast diversity in the ocean could be compromised signficantly. Estimates of marine microbial diversity are on the order of >20,000 microbes per liter of seawater, and likely, most of these species are not adapted to oily water.

Isolation of DNA from Oil Contaminated Soil and Water

To assist research on the impact of the oil spill on microbial diversity in soil and water from samples collected from the Gulf, MO BIO has created a webpage  for researchers who wish to share best practices.  The website is www.mobio.com/pages/oilspill.html.

Previous research using the UltraClean Soil DNA Isolation Kit and PowerSoil DNA Isolation Kit were very successful for isolation of microbial DNA from oiled water and soil because of the use of inhibitor removal technology. We are also sending samples of the PowerWater DNA Isolation Kit and PowerLyzer PowerSoil DNA Isolation Kit to oil spill researchers who contact us for help.  Researchers are welcome to send us any recommendations, protocols, or results they want to share with the community using any technique; we will post your feedback. If you have questions on how to work with oil spill samples, let us know and we can put you in contact with scientists who can offer assistance.

Volunteer for the Oiled Wildlife Care Network

There is another way to help with the oil spill and that is to contact a participating organization in the Oiled Wildlife Care Network (OWCN) and find out how to become a trained volunteer. In the event that an oil spill were to occur in California, or your coastline state, people are needed to become pre-trained on how to clean birds and rehabilitate animals. The OWCN webpage also contains information on what to do if you encounter an oiled animal as well as links to their facebook and blog page.

For those of us in San Diego or Los Angeles County, there are many possible locations to become trained in wildlife rehabilitation and care, even when there is not an oil spill.  OWCN volunteer coordinator, Kaiti Ferguson, at the UC Davis School of Veterinary Medicine,  recommends the non-profit volunteer organization called Project WildLife. With many locations around San Diego county, you can read about their volunteer programs and take an active role in taking care of hurt wildlife.

For information on volunteering in the Gulf states affected by the spill, contact numbers are found here at the Deepwater Horizon Response website.

Long Term Impact…

A solution to stop the leaking oil is expected by the end of July. However, it will take many years of scientific research before we will truly know what impact the oil has had (and is having) on all living species in the Gulf, including us.

The Spill Continues….

Q:  Recently your group published a paper in PNAS where you demonstrated it is possible to link individuals to objects that they have touched by comparing the bacterial community on hands and computer keyboards.  What led you to conduct this study?

A:  In earlier work, we looked at the diversity of bacteria living on different people’s hands, how it changed over short periods of time and what happened to bacterial communities after hand washing and treatment with Ethanol.  Surprisingly we discovered that every person we tested had a unique bacterial community that would re-establish itself within 2 hours of hand washing.  Even when hands were washed with Ethanol, those bacteria would regrow relatively quickly without much change.  Given the resilience of these bacteria we surmised that those bacteria would likely transfer to objects that are frequently touched by an individual. 

Q:  How foolproof is the idea that bacterial communities are completely unique to individuals?  Are there commonalities between members of families living in the same house or between parents and children? 

A:  Further work needs to be done to determine how relationships and proximity impacts these bacterial communities.  However, all of our current work says that most people’s bacterial communities are fairly unique.  Currently we’re evaluating the microbiomes of relatively healthy volunteers.  But eventually we hope to link changes and differences in human bacterial communities to disease states.

Q:  Can personal bacterial communities change over time?

A:  Currently we’re evaluating the microbiomes of relatively healthy volunteers at single timepoints.  Our future projects will include a wider demographic of volunteers from more age groups and health backgrounds.  Eventually we hope to link changes and differences in human bacterial communities to disease states, such as colon cancer, Irritable Bowel Syndrome and Crohn’s disease. 

Q:  How have MO BIO’s products assisted you in doing this work?

A:  MO BIO’s PowerSoil kit has an extremely high success rate for PCR.  95% of DNA samples extracted with this kit just work in our PCR reactions.  Most other kits have a 50% success rate.  We use this kit for all of the samples that we process.  Also, because bacterial DNA is so prevalent, we find that the Certified DNA-Free PCR water is key to preventing non-specific amplification in our reactions.

In this video, Dr. Fierer and Dr. Lauber talk about their work on microbial communities on skin:

For more information and a complete publication list, please visit the Fierer’s lab website:  http://www.colorado.edu/eeb/EEBprojects/FiererLab/index.html

Related papers include: 

Dominguez-Bello, M.G., E.K. Costello, M. Contreras, M. Magris, G. Hidalgo, N. Fierer, R. Knight. In Press. Delivery mode shapes the acquisition and structure of the founder microbiota across multiple body habitats in newborns. Proc. Natl. Acad. Sci. USA. Jun 2010; 10.1073/pnas.1002601107.

Fierer, N., C.L. Lauber, N. Zhou, D. McDonald, E.K. Costello, R. Knight. 2010. Forensic identification using skin bacterial communities. Proc. Natl. Acad. Sci. USA. 107: 6477-6481. 

Costello, E.K., C.L. Lauber, M. Hamady, N. Fierer, J.I. Gordon, R. Knight. 2009. Bacterial variation in human body habitats across space and time. Science. 326: 1694-1697

Fierer, N. M. Hamady, C.L. Lauber, R. Knight. 2008. The influence of sex, handedness, and washing on the diversity of hand surface bacteria. Proc. Natl. Acad. Sci, USA. 105: 17994-17999.

Bead beating is a great way to do what enzymes take hours to accomplish and sometimes never fully succeed in, which is cell lysis to release DNA or RNA for isolation. While enzymes can be successful for DNA isolation from a limited number of sample types, results are achieved a lot faster if you break down the matrix first. And RNA cannot be isolated in a timely fashion without the use of some kind of mechanical maceration.

The questions inevitably arise though, how hard do I need to beat to lyse my sample and how do I know what bead type to use?  The answers depend on a great number of variables, so to avoid beating your head against the wall to sort through them all, I have written this two-part blog series offering advice on the methods that we have used at MO BIO and found to work best for us.

This first article will focus on guidelines for RNA from tissues and plants.  There is so much to discuss about soil and microbes, so we’ll keep that for the second blog article, which will include data from our own research.

The Old Way to Isolate RNA- Liquid Nitrogen

RNA from tissues always requires serious pulverization. In the past, the most common method was using liquid nitrogen to freeze the sample and a mortar and pestle to grind the tissue to a powder. Although this approach works well, it is not complete. Once the sample is powdered and resuspended in a chaotropic lysis buffer, the genomic DNA is still high molecular weight and will add viscosity to the sample that can clog spin filters. To overcome this, the next step is to shear the genomic DNA with a needle and syringe which improves the efficiency of removing the genomic DNA from the column.

Drawbacks of Liquid Nitrogen Processing and Rotor Stator Homogenizers

Now, however, it’s the year 2010 and liquid nitrogen/ mortar and pestles are not the preferred method. Using these outdated methods, you need to either clean tools between samples, or you need to have a lot of them on hand and ready for use. The same is true for hand held rotor-stator homogenizers. This method is excellent for breaking a tissue down quickly and thoroughly so that the RNA is isolated with minimal degradation. However, the probe also needs to be cleaned between samples and there is always risk of cross-contamination. If you can use disposable probes on your rotor-stator, it is a better way to go. The drawback, however, is that you still can only process one sample at a time.

High Velocity Bead Beating- More Samples, No Cross-Contamination

This is where the high powered bead beaters come in and supersede the abilities of one-at-a-time methods. At MO BIO, we’ve developed a new instrument called a PowerLyzer™ bench top homogenizer. After working with and testing many models, we custom designed a machine with features that had everything we wanted for our own use, knowing that everyone else using bead beaters would desire the same changes. Basically, it’s a lot quieter and doesn’t vibrate your entire bench top.  And, the homogenization time needed for best results is also shortened (resulting in less heat and damage to the nucleic acids) because the horizontal positioning of the bead tube, (similar to the vortex adapter), is more efficient at grinding.   Less time means better RNA integrity.

What Bead Tubes do I Use?

For RNA from plant and animal tissues, we use the 2.8 mm ceramic bead tubes for two reasons. First, the 2.38 mm bead size is perfect for a 10-20 mg piece of animal tissue or 50 mg of plant tissue. We tested the 1.4 mm beads for tissues and the small size does not give as good a result with short homogenization times.  The longer time needed to liquify the sample increase degradation of RNA.

Using a single bead type (vs. a bead mix) allows for better consistency and homogeneity from prep to prep, since the larger more effective beads have complete access to the tissue and are not blocked by smaller less effective beads. The second reason for using the 2.8 mm ceramic bead tubes is because they are pre-loaded in a “Tough Tube” which is a specially made plastic bead tube that can withstand high force without breaking.

What is the Best Speed and Number of Cycles for Homogenization

This will vary based on the instrument used.  For animal tissues, we tested a wide range of speeds from 3500 to 5000 RPM, and while they all worked, the optimal RNA recovery was observed using 3500 RPM for 2 x 45 seconds using a 30 second rest between cycles.  This speed is about equivalent to a setting of 5.5 on the FastPrep. However, unlike the FastPrep, the PowerLyzer allows you to program any number of cycles and rest  time in between cycles, so you can save the protocol and program others and keep them handy for next time.

For Plant RNA, we found the optimal setting to be 4200 RPM for 2 x 45 second cycles with a 30 second rest in between cycles.  When 3500, 4200, and 5000 RPM were compared, 4200 gave the highest yield RNA from a variety of samples that included leaf, stem, roots, and seeds.

Summary

High powered bead beaters have many advantages over methods that process only one sample at a time. But for many people, this means re-optimizing current protocols. Fortunately, we’ve done a lot of the optimization already so you can get up and running right at the start. And RNA yields and integrity are going to be better when you can homogenize everything quickly and at once, and not have long lag times with your samples on ice while you move through all your preps.

If you are interested in ready-to-use kits for the PowerLyzer™ or other high powered bead beaters that are complete with the validated and optimal bead tube, you can find more information at these links.

For RNA from tissues go to the PowerLyzer UltraClean Tissue & Cells RNA Isolation Kit page and for RNA from plants, go to the PowerLyzer UltraClean Plant RNA Isolation Kit page. 

The PowerPlant DNA Isolation Kit is ready-to-use on high powered bead beaters, with tough tubes containing 2.38 mm stainless steel beads.

For DNA from soil and microbes, we will discuss this in an upcoming article and will present to you some very interesting data!

~Suzanne

Question:

 I am looking for suggestions for storing filters for subsequent DNA extraction using your PowerWater kit. We are heading out this weekend to the Gulf of Mexico and Florida Keys and will be collecting water samples for microbial analysis. We will be able to filter in the field but not complete the extraction process. Additionally our freezer space is limited. Do you have suggestions for storage of the filters that is not dependent on freezing. Is EtOH a possibility (will it work with the extraction process downstream?)  This is a last minute trip and we are trying to work with materials at hand as best we can.

 Thanks for your insight.

Answer from Heather Callahan, Ph.D.

Thank you for contacting us!  Here are some stabilization recommendations based on work I have been doing for DNA and RNA since last year. 

 These recommendations can be modified to work in Sterivex units or with flat filter membranes.  

Ethanol does not work.  Experiments using either 70% or 100% ethanol resulted in nucleic acids that are completely degraded and lost. 

Clean Water

Clean water samples can be stored dry in the kit bead tubes (or sterivex unit) at either room temperature or cold (refrigerated to -20 C).

Cold is usually best and DNA and most of the time RNA is very stable if the membrane is dry.  These results have been very consistent.  This is the easiest approach because if you are using the flat filter membranes, when you return to the lab, you can just add the lysis buffer and follow the kit protocol without having to transfer the membrane. With Sterivex, you can remove the membrane upon return and place into the PowerWater Bead Tube and begin the prep.

Dirty or Polluted Water

Filters from dirty water samples (muddy or heavily discolored) can be stored dry in the PowerWater bead tube also, but should be stored cold for best results. DNA from dirty samples appears to have the best integrity when the sample is stored at 4C vs. room temperature.  For short periods of time (3 days), room temperature storage may be ok and for long term storage, dry membranes chilled or frozen will result in high quality DNA.

We have also tested the use of LifeGuard Soil Preservation Solution for preserving the DNA and RNA from water filters. For dirty samples, this may be helpful to reduce degradation. Place the water filter into the bead tube and then use 2-4 ml of LifeGuard to saturate the filter. Storage at 4C or colder is still best for long term storage (> 1 week).

To process water filters that have been soaked in LifeGuard, centrifuge the bead tube containing the sample and beads to pellet all bacteria that may have fallen off the filter. Remove as much of the stabilization solution as you can so the lysis reagents are not diluted out significantly.

Alternatively, it would be better to store the filter in stabilizer without the beads so that the solution can be removed efficiently. If you need some empty PowerWater Bead Tubes before your next trip, let us know. If you plan to store the filters dry, then use the bead tube containing beads and then you can begin with the protocol as soon as you return to the lab.

Water Containing Metals

Just a side note regarding water that may be relatively clean from humic substances but contain high amounts of metals, such as water collected in acid mines or underground caves. Feedback from researchers has been to use the PowerWater DNA Isolation Kit.  Scientists have reported back to us that the method incorporating IRT has been successful at providing high quality DNA free of PCR inhibitors.  We are always collecting feedback so please let us know if you are working with metal contaminated water and what works best for you.

Summary

Since each project is different and often times you really have no information in advance about what your water will be like, you can call us to discuss and we can guide you further based on our experience. You may want to try a couple different methods to see which works best for you.

We are always looking for feedback from researchers using products so if you have any results of your own with water filter storage or if you want to try some comparison studies in collaboration with us, please contact us and let us know. 

And for processing DNA from microbes from oil, we have some references and recommendations depending on whether you are working with sediment or water filters. Contact us for advice.

We still have some of our PowerWater T-shirts left. Any blog readers working with the UltraClean Water DNA Kit or the PowerWater or RapidWater DNA Isolation Kits who would like to work with us to compile the results of water DNA purification from various sources of water and the best practices for stabilizing the DNA from those sources will receive one of our t-shirts.  Contact us at technical@mobio.com to discuss your next trip and the water type and filter type and we can provide advice and reagents.

For more information on How to Choose a Water Filter and for a video on how to transfer them from the filter funnel to the bead tube, click this link.

~Suzanne

We would like to send out some special thanks to researchers who collaborated with us to make this one of MO BIO’s best meetings ever.

Seminar presentation:

MO BIO would like to thank Dr. Janet Jansson for an excellent seminar Monday evening at the Hyatt. Her breadth of projects spanning from Crohn’s disease to the oil spill in the gulf makes one wonder if she ever sleeps. The seminar is available for viewing now.

Poster presentations:

We would also like to thank the labs that collaborated with us to generate data for posters we presented.

	Dr. Charles Lee, from the Craig Cary Lab, worked with us for months generating data from time course experiments and using soil collected from Antarctica to analyze the effects of LifeGuard Soil Preservation Solution on RNA isolation. It was a great body of work that was an incredible effort.
	Dr. Chris Kitts and Alice Hamrick helped us to ask some important questions about the differences in microbial populations isolated using different bead beating methods (vortex vs. the PowerLyzer 24 Homogenizer). Two undergraduates, Victoria Valencia and Mira Elnan, did numerous T-RFLP experiments to analyze the results from 6 different soils. In the end, we found that actually, bias seen with DNA extraction has a lot more to do with the soil than the DNA extraction method!
	MO BIO Labs presented a poster on a novel method for isolating DNA and RNA from biofilms. This project was a collaboration with several researchers around the globe working with various biofilms with ranging levels of difficulty in lysis and inhibitor content.
	And our final poster examined the quantitative effects of IRT on humic acid contaminated samples and the effects on both qPCR and RT-PCR.

It was a great experience to be at ASM and representing our scientific endeavors during the poster sessions with all of you, our customers. Thanks to everyone who came by our booth and posters.

How can you work with the MO BIO R&D team?

We were asked by a few people, how does one get the opportunity to collaborate with MO BIO and do a poster together?  It’s easy. Just contact me at technical@mobio.com or skennedy@mobio.com and we’ll discuss it.

We have questions we want to ask and are always looking for labs with undergraduates who need a cool project to do. Or if your student has an idea for a project and needs support and direction, we can help there too.

In addition, if you want to be involved in evaluating new methods for MO BIO and be on our beta-testing team, let us know. We always have new methods and ideas that need outside validation. And this is a great way to get your foot in the door for a career in biotech. By collaborating with a biotech company and learning the ropes of how we look at science and form questions from a commercial stand point, it will help make your resume much more attractive to industry hiring managers.

We hope to see you all at ASM 2011 in New Orleans, LA. Start planning now!

~Suzanne

I thought it would be a good time to review a human microbiota paper for all our readers. As an introduction to our guest speaker at our ASM Workshop on Monday, May 24th, this article discusses  Dr. Jansson’s 2007 study titled: “Molecular Fingerprinting of the Fecal Microbiota of Children Raised According to Differet Lifestyles“, from AEM, vol. 73, No.7, p.2284-2289.

Introduction:

The human intestinal community is very unique to each individual. While it doesn’t change much, with the exception of when antibiotics are used, the gut flora is quite stable and is heavily dependent on diet and lifestyle.

To gain a better understanding of the effects of diet and lifestyle on the human gut microbiota, this study looked at fecal microbial communities in children raised with two different types of diets. The first group of kids were living farm life,  consumming unpasteurized dairy products and home grown food and having frequent contact with farm animals.

The second group of kids were raised following an anthroposophic lifestyle, which means that their diet consisted of organically produced foods and vegetables were preserved by fermentation with lactobacilli. These children tended to be breast fed longer than average children. In addition, these kids did not receive antibiotics as much and their parents avoided vaccinations such as with MMR.

To determine the differences in the microbial populations of these groups of children who were between the ages of 5 and 13 years, the authors used T-RFLP analysis after isolation of the DNA from fecal samples using the UltraClean Soil DNA Isolation Kit combined with high powered bead beating. The fecal microbiota of 90 children who were either living on farms or attending Steiner schools (kids living the anthroposophic lifestyle ) were from Germany, Sweden, and Switzerland.

Methods:

Children did not have antibiotics during the last 3 months and were not ill at the time of sampling. Of the 90 children, 23 were anthroposophic kids, 19 were controls- kids living in the same area but attending public schools, 26 kids were living on farms and 22 control kids were not living on farms but in the same area.

DNA was isolated from fecal samples and T-RFLP performed for 16 s rRNA for total bacteria and also for lactic acid bacteria (LAB). The 16s rRNA genes were also cloned and sequenced to confirm identities of bacterial species that dominated in some of the children.

Results:

A total of 140 different TRFs were identified and each individual had a unique profile. Only a very small number of TRFs were common to all subjects. There was no clustering with regards to lifestyle, sex, diet, or origin. Of the predominant TRFs, the genera Eubacterium and Clostridium were the most common (90/90 and 89/90) respectively.

The Steiner children had more diversity in their fecal bacteria compared to farm children while the farm children had higher abundancies of some TRFs. Based on the data and questionnaires, the consumption of organically produced food correlated with high diversity while high consumption of farm milk correlated with low diversity. Children who never used antibiotics had higher diversity, as expected, while children who received the MMR vaccine showed lower diversity.

Using the LAB primers to assess the differences in commensal gut microbiota, one of the dominant clones, TRF 250, was unclassifiable below the phylum level, with the closest match to Clostridium ramosum (88%). This clone appeared in 57/82 individuals in the Steiner children and the reference groups but not the farm children. This particular phylotype may play an important role in the gut microbiotic environment given its dominance in the higher gut diversity children.

Conclusions:

Every individual has their own very specific gut microbiome.  No two samples are alike.  However, there are some predominat species that may be higher or lower in individuals based on diet.

This study found that a difference existed in the gut microbiota of children based on their lifestyle which did not relate to the geographic origin, as some other studies have observed. The Steiner children, who were from Sweden, Germany, and Switzerland, ate organically grown foods and foods fermented with lactobacillus as a preservative, had a higher diversity of bacteria. These kids in general do not get vaccinated and avoid antibiotics. The farm raised children had lower diversity but higher overall abundancies of gut microbes.

Is high diversity better? Higher diversity of gut microflora is considered better because it allows for better resilience in fighting infection or during disturbances.  For example, it has been previously reported that patients with Crohn’s disease have lower gut microbial  diversity, suggesting a link to human health. In the present study, farm children had the lowest diversity although they have also been shown to have a lower rate of developing allergies, supporting the hypothesis by Ley, RE, et al (2006), that “keystone species” can alleviate the requirement for higher diversity.

Of the Lactobacillus biota, one clone of interest, TRF 250, was predominant in all the kids except the farm raised children. This sequence does not match with any significant similarity to any known organism below the phylum level.  More clarity on the phylogeny and function of this organism could lead to more answers in the search for understanding the difference in human intestinal health.

Summary:

This interesting research demonstrates how much more we can know and ask about the human microbiome. Every discovery leads to many more questions.  The identification of healthy and disease gut microbiota over the next decade of microbiology research will allow us to understand the cause and effects of many diseases, including colon cancer. I am looking forward to hearing about the progress and interesting finding by many more of the human microbiome researchers this year at ASM 2010.

~Suzanne

See you at ASM Booth 1020. Come chat with us over food and drinks at our workshop at ASM Monday evening, the 24th. We look forward to meeting you and hearing your thoughts on what we can do better and how we can aid your research.

Remember MO BIO in your Methods Section of your Poster

If you haven’t received a MO BIO t-shirt yet then here is your chance!  Simply mention which MO BIO product you used in the methods section of your poster or during your talk and we will give you one of our new Biofilm t-shirts. 

Send us your abstract and poster details (location, date, time) in advance and take advantage of the opportunity to be included in the MO BIO Poster Guide to ASM.  Don’t miss the chance to be featured on our website and at our booth!

We’ve also planned a lovely evening including food and drinks….

In addition to networking with you at the booth and posters, MO BIO is hosting an event that we are sure will be enjoyed by all.  Janet Jansson, will be speaking on her work and the importance of DNA quality on Monday evening at the Grand Hyatt.

Please be our guest and listen to Dr. Jansson speak on:

Yield, Quality, and Purity: Considerations for DNA Extraction from Complex Microbiological Samples
Janet Jansson, Lawrence Berkeley National Labs
Introduction by Suzanne Kennedy, MO BIO Labs

When: Monday, May 24, 2010

Where: Room BIO5 Auditorium
Manchester Grand Hyatt Convention Center
1 Market Place
San Diego, CA 92101
7:00 –9:00 pm

Refreshments: Hot hors d’oeuvres and a cocktail will be provided, along with an open cash bar for your convenience

Raffle: All attendees will be entered into a drawing for a chance to win a copy of the book Environmental Molecular Microbiology, published by Horizon Press, and co-edited by Dr. Jansson and Dr. Liu.  Attendee must be present to be entered to win.

Register now: Seating is limited and pre-registration is advised. Email info@mobio.com with your name and contact information and put “ASM Seminar” in the subject line to guarantee your attendance at Dr. Jansson’s seminar today.

Abstract:  Extracting nucleic acids with high yields and purity from microbial samples can be extremely challenging. Complex environments such as soil, biofilms, and gut exacerbate the problem  because microorganisms in these conditions are tough to lyse and include high levels of enzymatic inhibitors that co-purify with nucleic acids.    

Likewise, successful microbial community analysis depends on high efficiency extraction and purification to accurately reflect the quantity of DNA or RNA in a sample. Extreme differences in environmental samples may exist between neighboring collection sites, collection depths, or even between individual gut microbiota, increasing the challenge of accurately profiling these ecological habitats. In this workshop, we will discuss considerations for obtaining DNA with high and accurate yields and purity from various samples isolated from environmental sources. Popular misconceptions to watch for when analyzing the DNA will be addressed. Dr. Jansson will present her work focusing on the analysis of microbial ecology of diverse sample types and the importance of DNA yield, quality, and purity in research.

About the speaker:

Dr. Jansson received her Ph.D. in Microbial Ecology from Michigan State University in the laboratory of Jim Tiedje. She spent 20 years in Sweden, starting with her postdoctoral research at Stockholm University. She was a Professor of Microbiology at Södertörn University College and then Professor (Chair) of Environmental Microbiology at the Swedish University of Agricultural Sciences (SLU). She was also Vice Dean of the Faculty for Natural Resources and Agricultural Sciences at SLU. She is currently a Senior Staff Scientist in the Earth Sciences Division at Berkeley National Labs and a Chief Editor of  The ISME Journal.  Her expertise in molecular microbial ecology and “omics” approaches focuses on soil, marine sediment, and the human gut environments.

MO BIO Posters at ASM:

MO BIO scientists are proud to have four posters accepted in the 2010 ASM annual meeting. Below are the details of our posters. Let us know how we can find you!

1. Abstract Title:Taking Snapshots of Microbial Communities: RNA-Based Assessment of Soil Microbial Communities Stored in a Novel Bacteriostatic RNA Preservation Solution
Authors:  C. K. Lee¹, S. J. Kennedy², V. J. Moroney², M. N. Brolaski², S. C. Cary^1,3;
¹Univ. of Waikato, Hamilton, NEW ZEALAND, ²MO BIO Lab., Carlsbad, CA, ³Univ. of Delaware, Lewes, DE.
Session Title: Soil Microbiology: Other
Session Number: 153/N
Poster Presentation Date/Time: Tuesday May 25, 2010 10:45 AM – 12:15 PM
Poster Presentation Number: N-1345
Poster Board Number: 659

2. Abstract Title:Comparison of Microbial Populations Isolated from a Variety of Soils using Different Homogenization Methods During DNA Extraction
Authors:  N. von Atzigen¹, V. E. Valencia², M. R. Elnan², M. J. Bunnell², M. W. Black², S. J. Kennedy¹, M. N. Brolaski¹, C. L. Kitts²;
¹MoBio Lab., Carlsbad, CA, ²California State Polytechnic Univ., San Luis Obispo, CA.
Session Title: Soil Microbiology: Other
Session Number: 153/N
Poster Presentation Date/Time: Tuesday May 25, 2010 10:45 AM – 12:15 PM
Poster Presentation Number: N-1342
Poster Board Number: 656

3. Abstract Title:  Development and Evaluation of a New Protocol for DNA and RNA Isolation from Microbial Mats
Authors:  H. A. Callahan, S. J. Kennedy, M. N. Brolaski;
MO BIO Lab., Inc., Carlsbad, CA.
Session Title: Environmental Biofilms
Session Number: 185/Q
Poster Presentation Date/Time: Tuesday May 25, 2010 1:00 PM – 2:30 PM
Poster Presentation Number: Q-1866
Poster Board Number: 894

4. Abstract Title:  Quantitative Assessment of the Removal of Humic Acid from Purified DNA and Environmental Samples Using Inhibitor Removal Technology
Authors:  H. A. Callahan, S. J. Kennedy, M. N. Brolaski;
MO BIO Lab., Inc., Carlsbad, CA.
Session Title: General Environmental Microbiology – II
Session Number: 287/Q
Poster Presentation Date/Time: Wednesday May 26, 2010 1:00 PM – 2:30 PM
Poster Presentation Number: Q-2855
Poster Board Number: 888

Q: What is the focus of your research at McMurdo?

A: Essentially, we are looking at the rock bottom of the food chain, low carbon oligotrophic microbes, with a particular focus on fungi that fix carbon. Antarctica is a perfect setting for these harsh and unique conditions in which the food chain is forced to ‘get creative’. Certain remote locations will be comprised of soil with no organic carbons present, suggesting that plants may never have been present. This offers a setting where microbes must adapt to utilizing nutrients and energy from volcanic rocks. In areas containing no vascular plants, you may still see a wide range of bacteria, but the fungal diversity is unlike any other on the planet. Incredibly, life even exists in the permanently ice covered Lake Bonney, ‘that’s why microbiology is so interesting!’.

Q: Can you describe the conditions while working in this environment?

A: The Antarctic environment is unlike any other on Earth, with extreme fluctuations. Conditions can change so rapidly, we have experienced a temperature drop by 30°C simply from a cloud passing by overhead. We were once surprised by a solar eclipse, which was only noticeable by the dramatic temperature drop! The land can seem like a completely foreign planet. Some of our expeditions will involve walking 15 to 20 miles per day, through areas of high iron content, with eerie Mars-like land. Although McMurdo is at sea level, is has an effective altitude of 1,000 feet. On our last climb up to the caves at Mt Erebus, we stopped at the edge of Fang Glacier for three days of acclimitization. Just as we were preparing to move on, we became engulfed by a horrendous storm, stuck inside our tents for seven days. This was over Thanksgiving, with a feast consisting of one cup of pea soup and a few crackers. It was an adventure to say the least.

Q: What is the significance of your research?

A: The conditions present at our sites, while unlike most currently on Earth, have been compared to conditions on other planets, and most notably to those present on early Earth. Discovering what species are able to survive and ultimately dominate in this harsh environment, as well as exactly how they utilize the nutrients present, converting inorganic compounds to organics, may provide a substrate for the evolution of early life.

Q: What are your plans for the next upcoming endeavour?

A: Our next trip will be a follow up to the previous. We will be removing basalt traps at glacial dive holes for our next collection of data. We will also be headed to Blood Falls, named after its appearance due to trapped marine containing high iron content for diving and cave work. Also, we will travel to Walcott glacier, which is much further south, to compare and contrast the latitude and chemical gradient.

Q: How have MO BIO kits helped in your research?

A: We have relied on a range of MO BIO kits including the RNA PowerSoil® Total RNA Isolation Kit and UltraClean® Mega Soil DNA Isolation Kit to overcome common inhibitors contained within these challenging samples.

For more information on Dr. Laurie Connell’s research, please visit the Connell Lab homepage at http://www.umaine.edu/nunatak/index.htm