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BiOstic® Bacteremia…Our Dirty Little Secret

Apr 10, 2015
Michelle Tetreault Carlson

bacteriaInBlood

When the BiOstic® Bacteremia DNA Isolation kit was originally designed, it was optimized for the extraction of bacterial DNA from infected blood. However, it has turned out to be useful for so much more. Bacteremia is the presence of bacteria in the blood and under normal circumstances blood should be sterile, but with the insertion of a catheter into the body or through an open wound, bacteria can gain entry and take hold.

Even a small numbers of bacteria can be a real problem and make people very sick. Hospitals need to have a low bacterial detection threshold, and in order to achieve this the blood is first cultured, which can take 48 hours and is applicable only to culturable microbes. To complicate things further, some patients have already been dosed with antibiotics or other drugs which can interfere with the culturing process.

The basic premise of the BiOstic® Bacteremia DNA Isolation Kit is to isolate DNA from a small number of bacteria inside a messy mixture of blood and anticoagulants or growth media. Standard blood or microbial kits won’t work for this. Microbial cells are harder to lyse than blood cells and the heme and lipids in the blood can be potent PCR inhibitors which need to be removed using specialized chemistry.

The BiOstic® Bacteremia DNA Isolation kit combines bead beating and detergent for complete bacterial cell lysis with MO BIO’s patented Inhibitor Removal Technology®, for the removal of heme molecules and other inhibitors from the DNA. The first steps in the BiOstic® Bacteremia DNA protocol are to remove a portion of cultured blood; centrifuge to pellet all of the cells and then bead beat the cell pellet with a strong detergent-based lysis buffer. The kit uses 2 ml MicroBead tubes containing fine garnet sand and is ideal for lysing microbial cells. Bead beating is followed by MO BIO’s Inhibitor Removal Technology®.This is the same powerful chemistry that is used in our soil and fecal kits and will remove all PCR inhibitors including heme, lipids, polysaccharides, humic acid, and heparin.

New Applications

Isolating microbial DNA from cultured blood is a pretty specific application. Soon after the BiOstic® Bacteremia DNA Kit was released, we found other situations where it was useful to get DNA from a dirty cell pellet. Customers started trying it on all kinds of other samples and it turned out to work fabulously. We thought it time to let you in on our dirty little secret.

Dirty Swabs

Swab samples which contain PCR inhibitors (fecal, vaginal, wound, intestinal/stomach, environmental surfaces) tend to contain a relatively small number of cells in a hot mess of PCR inhibitors. The BiOstic® Bacteremia Kit’s strong lysis buffer combined with its one step Inhibitor Removal Technologyâ turns out to be ideal for maximizing the DNA yields. To use a swab with this kit, start with the following protocol:

  1. Add 450 µl of the CB1 buffer directly to the MicroBead tube.
  2. Place and rotate the swab in the buffer and let it soak for a few minutes to release the cells into the solution.
  3. If the swab has a head that can be snapped off, go ahead and do so, leaving the swab in the tube. Otherwise remove the swab now while gently squeezing it against the wall of the tube to remove as much of the solution as possible.
  4. Proceed with the 70 C heating step and the rest of the normal protocol.

Dirty Microbial Pellets

Any situation where bacteria can be pelleted from a dirty (containing PCR inhibiters) liquid can work well with the BiOstic® Bacteremia DNA Isolation kit. Some examples include sputum, saliva, washes from dirty surfaces (nasopharyngeal and colorectal), and mixed culture isolates. To utilize these types of samples with this kit, follow this protocol:

  1. Centrifuge the liquid at 13,000 xg for 2 minutes to pellet the cells. The volume of liquid will depend on the sample. Aim for a wet cell pellet weight of 25 mg or less.
  2. Remove the supernatant.
  3. Resuspend the cell pellet in 450 µl of the CB1 buffer and add to the MicroBead tube.
  4. Proceed with the 70 C heating step and the rest of the normal protocol.

DIRECT FROM (uncultured) BLOOD

Customers often want to avoid culturing blood because of the time and possible bias involved. The kit can also be used for the isolation of bacterial DNA directly from blood without culturing, but less starting sample has to be used in order to avoid clogging of the spin column. To use the kit for DNA isolation from whole blood, follow this protocol:

  1. Centrifuge 50-500 µl of whole blood at 13,000 xg for w minutes to pellet all of the cells.
  2. Pipette off the supernatant and dispose of as hazardous waste.
  3. Resuspend the cell pellet in 450 µl of the CB1 buffer and add to the MicroBead tube.
  4. Proceed with the 70 C heating step and the rest of the normal protocol.

And More…

In addition to the sample types mentioned above, the BiOstic® Bacteremia Kit has been used for filtered plant washes, feces and even raw milk. We’ve listed some recent references below. Maybe you’ll come up with more ideas.

So what started as a bacteremia kit is now much more – should we ask the Marketing Team to rename this product: PowerDirtyLittleSecret? Let us know you opinion!

References

Direct from Blood:

Identification of Different Bartonella Species in the Cattle Tail Louse (Haematopinus quadripertusus) and in Cattle Blood
Ricardo Gutiérrez, Liron Cohen, Danny Morick, Kosta Y. Mumcuoglu, Shimon Harrus, and Yuval Gottlieb
Appl. Envir. Microbiol., Sep 2014; 80: 5477 – 5483.

From Cultured Infected Tissue:

Mupirocin and Chlorhexidine Resistance in Staphylococcus aureus in Patients with Community-Onset Skin and Soft Tissue Infections
Stephanie A. Fritz, Patrick G. Hogan, Bernard C. Camins, Ali J. Ainsworth, Carol Patrick, Madeline S. Martin, Melissa J. Krauss, Marcela Rodriguez, and Carey-Ann D. Burnham
Antimicrob. Agents Chemother., Jan 2013; 57: 559 – 568.

A Serologic Correlate of Protective Immunity Against Community-OnsetStaphylococcus aureus Infection
Stephanie A. Fritz, Kristin M. Tiemann, Patrick G. Hogan, Emma K. Epplin, Marcela Rodriguez, Duha N. Al-Zubeidi, Juliane Bubeck Wardenburg, and David A. Hunstad
Clinical Infectious Diseases, Jun 2013; 56: 1554 – 1561.

Direct from Swabs:

Rectal Swabs Are Suitable for Quantifying the Carriage Load of KPC-Producing Carbapenem-Resistant Enterobacteriaceae
A. Lerner, J. Romano, I. Chmelnitsky, S. Navon-Venezia, R. Edgar, and Y. Carmeli
Antimicrob. Agents Chemother., Mar 2013; 57: 1474 – 1479.

Extravaginal Reservoirs of Vaginal Bacteria as Risk Factors for Incident Bacterial Vaginosis
Jeanne M. Marrazzo, Tina L. Fiedler, Sujatha Srinivasan, Katherine K. Thomas, Congzhou Liu, Daisy Ko, Hu Xie, Misty Saracino, and David N. Fredricks
The Journal of Infectious Disease, May 2012; 205: 1580 – 1588.

Filtered plant washes:

Ecological Succession and Stochastic Variation in the Assembly ofArabidopsis thaliana Phyllosphere Communities
Loïs Maignien, Emelia A. DeForce, Meghan E. Chafee, A. Murat Eren, and Sheri L. Simmons
mBio, Jan 2014; 5: e00682-13.\

Direct from Saliva:

Salivary Microbiota and Metabolome Associated with Celiac Disease
Ruggiero Francavilla, Danilo Ercolini, Maria Piccolo, Lucia Vannini, Sonya Siragusa, Francesca De Filippis, Ilaria De Pasquale, Raffaella Di Cagno, Michele Di Toma, Giorgia Gozzi, Diana I. Serrazanetti, Maria De Angelis, and Marco Gobbetti
Appl. Envir. Microbiol., Jun 2014; 80: 3416 – 3425.

Direct from feces:

Membership and Behavior of Ultra-Low-Diversity Pathogen Communities Present in the Gut of Humans during Prolonged Critical Illness
Alexander Zaborin, Daniel Smith, Kevin Garfield, John Quensen, Baddr Shakhsheer, Matthew Kade, Matthew Tirrell, James Tiedje, Jack A. Gilbert, Olga Zaborina, and John C. Alverdy
mBio, Sep 2014; 5: e01361-14.

Direct from Raw Milk:

“Remake” by High-Throughput Sequencing of the Microbiota Involved in the Production of Water Buffalo Mozzarella Cheese
Danilo Ercolini, Francesca De Filippis, Antonietta La Storia, and Michele Iacono
Appl. Envir. Microbiol., Nov 2012; 78: 8142 – 8145.

Blood Culture:

Herbaspirillum Species Bacteremia in a Pediatric Oncology Patient
Edward D. Ziga, Todd Druley, and Carey-Ann D. Burnham
J. Clin. Microbiol., Nov 2010; 48: 4320 – 4321.

 

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Become BFFs With Your FFPE

Apr 02, 2015
Michelle Tetreault Carlson

Formalin-Fixed, Paraffin-Embedded (FFPE) tissues, the most common tissue preparation method for archiving bio-specimens, can be a valuable resource for genetic studies. The fixation process makes it possible for samples to be stored for years at room temperature, for analysis even decades later.

Chemical fixatives like formalin (a mixture of formaldehyde and methanol) preserve the structural integrity and morphology of the tissue by cross-linking neighboring amino groups between proteins. This traps other molecules like carbohydrates, lipids and nucleic acids in place.

While the fixation process preserves the structural integrity of the cells, it also denatures proteins and causes the degradation of RNA and DNA. The longer the fixation process and the age of the tissue the more damaged the nucleic acids.  As a result, the size of the nucleic acid fragments is generally small, in the 100-500 bp range. All this makes the extraction, amplification and analysis of nucleic acids a bit tricky. However, using appropriate protocols and kits it’s still possible and highly worth the effort.

MO BIO has two awesome kits for isolating DNA or RNA from formalin fixed paraffin embedded (FFPE) tissue. The traditional method for removing wax from FFPE tissue has always been to use xylene, a highly flammable and toxic organic solvent. The tissue is washed several times in xylene to dissolve the wax and then the xylene is removed by performing multiple washes with ethanol before doing the DNA isolation. This results in lots of extra handling where the tissue is repeatedly washed. Each time a wash occurs; some of the sample can be lost along with the wax and the solvent.

The BiOstic® FFPE DNA and RNA kits avoid such harsh treatments by leaving the wax intact for the extraction. By using an optimal combination of denaturing buffers and higher temperatures, the activity of proteinase K can be optimized so that complete digestion of the tissue occurs while the wax is melting. This reduces handling time and loss of tissue.

In this example (provided by a customer) using 10 micron thick single slices removed from histology slides, the samples in blue were pre-processed with xylene and DNA isolated following the manufacturer’s protocol and the BiOstic FFPE DNA samples were extracted. Yields were quantified on the Nanodrop.

In this example (provided by a customer) using 10 micron thick single slices removed from histology slides, the samples in blue were pre-processed with xylene and DNA isolated following the manufacturer’s protocol and the BiOstic FFPE DNA samples were extracted. Yields were quantified on the Nanodrop.

Both kits follow similar protocols.1 to 5 slices of FFPE tissue (or up to 15 mg) are digested with Proteinase K at low heat in order to liquefy the tissue and release the DNA.  Then a second heating step at higher temperature is used to remove cross-links that can inhibit PCR or other enigmatic reactions.

Because RNA degradation can be more severe for these sample types, the BiOstic® FFPE RNA Isolation Kit uses a gentler, pH neutral lysis buffer and the Proteinase K digest and the cross-link removal are done at lower temperatures and for shorter times.  We also added our new low elution silica spin filter with a final elution volume of only 20 µl, to this kit in order to increase the final RNA concentration.

Comparative analysis of the BiOstic® FFPE Tissue RNA Isolation Kit (MB) and competitors Q and LT of RNA extraction from a single 10 micron tissue slice of FFPE Normal Human Liver tissue. a)  1.2% TAE gel showing higher yields of intact RNA obtained when sample was prepared with the BiOstic® FFPE Tissue RNA Isolation Kit. b) Invitrogen Qubit™ Fluorometer readings show that higher yields of RNA were obtained when sample was prepared with the BiOstic® FFPE Tissue RNA Isolation Kit (MB).

Comparative analysis of the BiOstic® FFPE Tissue RNA Isolation Kit (MB) and competitors Q and LT of RNA extraction from a single 10 micron tissue slice of FFPE Normal Human Liver tissue. a) 1.2% TAE gel showing higher yields of intact RNA obtained when sample was prepared with the BiOstic® FFPE Tissue RNA Isolation Kit. b) Invitrogen Qubit™ Fluorometer readings show that higher yields of RNA were obtained when sample was prepared with the BiOstic® FFPE Tissue RNA Isolation Kit (MB).

FFPE tissues are a unique sample type with a lot of challenges, but when it comes to DNA or RNA isolation, we’ve made that part easy.

Samples are available and can be ordered on the web or upon request at no charge if you call/email customer service. If you have more questions such as how to remove C/T artifacts that could occur at a very low rate in some tissue, technical support is here to help. We’re waiting to help you out!

Some recent references for the BiOstic® FFPE DNA Isolation kit:

TERT promoter mutations are associated with distant metastases in papillary thyroid carcinoma
Greta Gandolfi, Moira Ragazzi, Andrea Frasoldati, Simonetta Piana, Alessia Ciarrocchi, and Valentina Sancisi
Eur. J. Endocrinol., Feb 2015; 172: 403 – 413.

Microbial communities present in the lower respiratory tract of clinically healthy birds in Pakistan
Muhammad Zubair Shabbir, Tyler Malys, Yury V. Ivanov, Jihye Park, Muhammad Abu Bakr Shabbir, Masood Rabbani, Tahir Yaqub, and Eric Thomas Harvill
Poultry Science, Feb 2015; 10.3382/ps/pev010.

Anaplastic Lymphoma Kinase–Positive Large B-Cell Lymphoma: Description of a Case With an Unexpected Clinical Outcome
Magda Zanelli, Riccardo Valli, Isabella Capodanno, Moira Ragazzi, and Stefano Ascani
International Journal of Surgical Pathology, Feb 2015; 23: 78 – 83.

 

 

 

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Whatcha Been Up To?

We are starting a new series of blogs that will include a short synopsis of a recently published peer-reviewed paper. We want to keep you (and ourselves!) up to date on the latest and greatest science news.

Psst, don’t forget about our Published Reference program, you get a free kit for sending us (reference@mobio.com) your published paper using our kits!  Not a bad gig!

mobio-blog-emelia

This month we picked:

Metagenomic Analysis of the Airborne Environment in Urban Spaces

Nicholas A. Be & James B. Thissen & Viacheslav Y. Fofanov & Jonathan E. Allen & Mark Rojas & George Golovko & Yuriy Fofanov & Heather Koshinsky & Crystal J. Jaing

This group collected samples by filtering air that trapped airborne microorganisms from urban spaces in Washington, DC. They extracted the nucleic acids off the filters and then used next-gen sequencing to characterize the community. This work is increasingly relevant in recent times of bio-terrorism. It is therefore very important to be able track this kind of activity and their paper describes a method to do so.

biohazard2

What’s even more interesting is that their experimental design included areas that had been purposely colonized by spore forming Bacillus thuringiensis serovar kurstaki, a known pesticide against gypsy moths. B. thuringiensis is non-pathogenic to humans but because it is so closely related to Bacillus anthracis, the agent that causes anthrax, it can act as a “pathogen surrogate” to determine the detection of bio-terrorism.

The bottom line is that they found a seasonal pattern (spring, summer, fall, winter) in the data and there was sufficient detection of the spore-forming organism. Therefore, they created a protocol that can be used for detection of potential aerosolized bio-terrorism activities.

Thanks for using MO BIO and please keep up the awesome science work, we love to learn and be inspired!!

 

 

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The Origin of Life on Earth and Aliens

Mar 02, 2015
Emelia

Hello MO BIO! IMG_3866

My name is Jesse McNichol and I’m a graduate student in the MIT/Woods Hole Joint Program and along with my supervisor, Dr. Stefan Sievert, I study the microbiology of deep-sea hydrothermal vents. These ecosystems are extremely unusual on Earth, since they are mostly supported by volcanic activity instead of the sun’s light. The oxidation of hydrogen sulfide and hydrogen gas are probably the ultimate source of food for all the teeming life around these hot springs, including the large fishes, tube worms and crabs.20141105135543

 

I’d seen these ethereal ecosystems before through a video monitor, and I was fortunate enough to see them up close and personal in during our research expedition in November 2014 from the window of the submarine Alvin! Seeing the huge black smoker chimneys and the giant tube worms reminded me why I became so fascinated with vents in the first place. Because they are so different and mostly independent of sunlight, many people (myself included) study them to better understand how life might exist on other planets, such as in the oceans of extraterrestrial moons Europa or Enceladus.

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Although a fascination with life on other planets is what brought me into this field, the motivations for this study are more ‘down to earth’. While the genomic revolution has given us an incredible amount of detail on which organisms exist in deep-sea vents, we actually know very little information about them. How fast do they grow? How much energy they need to survive? How do they interact with one another? In short, we have a very incomplete picture of the ecology of these unique deep-sea environments.

 

Being at the bottom of the ocean, under over 250 atmospheres of pressure, these ecosystems are naturally very difficult to sample. That’s why I’m fortunate to work at Woods Hole – not only do we have access to the R/V Atlantis and the submarine Alvin, my supervisor’s long-time collaborator Dr. Jeff Seewald has designed and pioneered a sampling device that can bring to the surface an accurate ‘snapshot’ of the deep-sea community, and keep the microbes under the intense pressure found in the deep sea. Although it had been used routinely before for chemical sampling, I was the lucky graduate student to get to try growing microbes for the first time in Dr. Seewald’s samplers.

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Given this incredible opportunity, I spent a lot of time thinking about what conditions to test to unlock the unsolved mysteries of these ecosystems. Eventually, I carried out 53 high-pressure incubations during two oceanographic cruises in 2014, which continue to yield new insights as I process the chemical and genetic data. In the end, this work will yield clear estimates on the productivity of the microbial community at deep-sea hydrothermal vents, which will help us understand the ecology of the whole system.

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For the general public, the scientific details will not be as important, but I hope that when people see how much more we have to learn about these ecosystems on Earth, it will inspire them to learn more about what I think is the greatest story yet to be uncovered – how life arose and evolved on Earth and how it could exist outside of our small planet.

IMG_1616

To support the telling of these stories, MO BIO plays a key role. Although I often still use ‘old school’ DNA extraction techniques, DNA and RNA extraction kits are essential for our everyday work in the lab. We use them to generate high-quality DNA and RNA for experiments that help us to understand these organisms and their ecology better, especially the effect of different chemical conditions on the activity and expression of poorly understood metabolic genes.

IMG_1620To read more about the November 2014 Research expedition click here for the official website and here for an article written about Jesse McNichol.

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Inspiring Women Scientists in Microbiology and Ecology: Part Deux

Feb 12, 2015
Emelia

There’s been some social media buzz lately about #WomenInScience. No better time than the present to update the list from our previous blog!

 

Inspired by #ISME15

Recipient of the ISME Young Investigators Award:

Ruth_LEY_2010_pic-2an4pef

Ruth E. Ley is an Assistant Professor of Microbiology at Cornell University in Ithaca, NY. She was trained in ecology and natural history at the University of California Berkeley (B.A.) and in ecosystem science and soil microbial ecology at the University of Colorado, Boulder, where she worked with Dr. Steve Schmidt (Ph.D.). Her post doctoral research was first with Dr. Norman Pace, working on highly diverse hypersaline microbial mats. She then transitioned to working with Dr. Jeffrey Gordon on the microbial ecology of obesity at Washington University School of Medicine. She is an author on 4 of the 10 most highly cited papers on the human microbiome. Her interdisciplinary group at Cornell works on the human microbiome at different scales of analysis, including large-scale genetic studies in human to discover novel pathways of interaction between host and microbiome, and mechanistic studies of those interactions using germfree mice as a tool to assemble and interrogate specific microbiotas. Dr. Ley’s awards have included the Hartwell Investigator Award, the Arnold and Mabel Beckman Young Investigator Award, a David and Lucile Packard Foundation Fellowship, and an NIH Director’s New Innovator Award.

Keynote Speaker at ISME:

Vorholt pic

Julia Vorholt is Professor of Microbiology at the Swiss Federal Institute of Technology Zurich (ETH Zurich), Switzerland. She carried out her Ph.D. work at the Max Planck Institute for Terrestrial Microbiology in Marburg, Germany. After a postdoctoral stay at the University of Washington, Seattle, USA, she returned to the MPI in Marburg as a group leader and subsequently headed a group at the Centre National de la Recherche Scientifique in Toulouse, France. Since 2006 Julia Vorholt is Professor at the Institute of Microbiology of ETH Zurich. She investigates how the environment, in particular the phyllosphere, shapes bacterial physiology, with an emphasis on metabolism, novel protein function and gene regulation. She applies metaproteogenomics to bacterial phyllosphere communities, uses synthetic bacterial communities to study microbe-microbe-plant interactions and develops Fluidic Force Microscopy for single cell analyses. She received the Otto-Hahn medal of the Max-Planck Society and is a member of the German National Academy of Sciences, Leopoldina.

Inspired by #MOBIO Customers:

allen-vercoe

Emma Allen-Vercoe

In her own words:

I began my research career with undergraduate and graduate studies at the Central Veterinary Laboratories (now Veterinary Laboratories Agency) and the Centre for Applied and Microbiological Research (CAMR, now the Health Protection Agency), UK, under the direction of Prof. Martin Woodward. There, I studied the enteric pathogen Salmonella enterica serovar Enteritidis, and developed a sound appreciation of the many obstacles that a enteric pathogen must overcome in the gut in order to cause disease. I became fascinated by the huge arsenal of virulence factors required by enteric pathogens in order to survive and proliferate in the gut environment.

I spent a brief postdoctoral period at CAMR, learning to work with technically challenging pathogens such as Mycobacterium tuberculosis and Campylobacter jejuni, before I relocated to Canada in 2001 to start a postdoctoral position at the University of Calgary, under the joint direction of Drs. Rebekah DeVinney and Mike Surette. Here I worked on Enteropathogenic and Enterohemorrhagic E. coli (EPEC and EHEC), using cell and molecular biology techniques to probe the fascinating interactions of their type III secretion systems with host cells.

I had always been interested in learning more about the normal microbial population inside the human gut, and in 2004 I was fortunate enough to win a Fellow-to-Faculty Transition award through the Canadian Association of Gastroenterology. This award allowed me to develop an independent research program aimed at the study of the normal human microbiota and its influence on human health and disease, a program that I brought with me to Guelph in December 2007.

My motto: “My microbes told me to do it”—->  (We at MO BIO are all going to start using that as an excuse all day everyday!)

My hobbies: Gardening, reading, reading about gardening

Thanks Ladies for all the inspiration!  We are honored at MO BIO to serve you…

Want to see more rockin #WomenInScience, #SciWomen, #WomenInSTEM, #GeniusWomen?

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Protect your RNA samples during DNase treatment

The DNase step is one of the most common causes of degradation or loss of the RNA during your extraction. DNase digestion is frequently performed on the spin column and although this can be a great way to save  time on the post extraction processing, it is not an efficient method for samples with large amounts of DNA (for example, spleen, thymus, and even some soils). In these cases, DNase digestion in solution is necessary.

The typical protocols for DNase involve inactivation of the enzyme using EDTA and heat. Both of these things can cause problems in RT-PCR. EDTA can inhibit the RT-PCR enzymes and heating the RNA can cause a reduction in integrity.  Additionally, most DNase enzymes are stored frozen and need to be aliquoted to avoid freeze/thaw cycles that can reduce enzyme efficiency.

We came up with a better system that protects the RNA all the way to the final step: DNase Max.

DNase Max is a liquid room temperature stable DNase with very high activity (1 ul of enzyme can digest 30 ug of DNA in 20 minutes). The best part is the clean up step. The DNase comes with a highly specific removal resin that binds the enzyme and cations and pulls them out of the RNA sample making it ready to use in qRT-PCR without any inhibitory additives or heat steps.  The resin is so efficient that completely removes 10 units of enzyme in the reaction, (figure below, lanes 3-4) compared to an alternative resin method incapable of removing the just 2 units of DNase enzyme used (lanes 1-2).

This means that you can protect your precious RNA as well as hours of work invested and get better accuracy in gene expression assays.

DNase MaxRemoval Resin completely removes DNase. Samples were subjected to DNase treatment and enzyme removal using the  DNase Max™ Kit or a competitor’s kit, and then analyzed for residual DNase activity using the MO BIO DNase-free certification assay. Lane 5 is the negative control and did not receive DNase. Samples were incubated for 1 hour at 37C, followed by inactivation for 5 minutes at 65C. Results are shown on a 1% agarose gel. The DNase Max Removal Resin successfully removed the DNase (lanes 3-4), while the competitor’s resin failed to remove all of the DNase from the samples (lanes 1-2).

Other tools to protect your RNA.

Isolation of RNA, no matter what the source, is nerve wracking, but especially when samples are limited or irreplaceable.  Because RNA is so labile, working quickly but carefully is the key. There are ways to protect your RNA during the procedure so that you can work at a relaxed pace and without so much anxiety.

The use of BME, consistent and fast homogenization, and RNase-free DNase with removal resin will be your ticket to success in every prep no matter what the sample. Certified RNase-free gloves are a great extra to have as well as UltraClean Lab Cleaner for removal of nucleases from the bench and equipment. We use these routinely in our labs.

 

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Vampire Ventures

Oct 30, 2014
Michelle Tetreault Carlson

15094287_s

Two vampires walk into a bar and call for the bartender.

“I’ll have a glass of blood,” says one.

“I’ll have a glass of plasma,” says the other.

“Okay,” replies the bartender, “That’ll be one blood and one blood lite.”

(insert laughter here)

 

Yep, we are celebrating the scary season and launching our new PowerMag Blood DNA/RNA Isolation kit!

Blood:  What’s in it?

Blood contains a mixture of plasma, red and white cells and platelets.  It is a unique beast among sample types, because while the quantity of nucleic acids in blood is copious, this genetic gold mine bathes inside a complex soup of cellular debris and protein. These contaminants can interfere with downstream PCR and sequencing.  It is the hemoglobin in particular, within the red cells, which causes major issues in DNA/RNA contamination and PCR inhibition.

Fortunately, dirty samples don’t scare us at here at MO BIO.  After all, we know how to get clean nucleic acids from feces and soil, no problemo!

Blood Collection and Storage:

Blood should be collected into an anticoagulant coated tube.  Otherwise, the blood will clot and bind up most of your DNA/RNA containing cells.  Standard blood collection tubes typically contain EDTA or citrate to prevent this.  Neither of these interferes with downstream genetic analysis.  Heparin can also prevent clotting but because it tends to bind to DNA, we don’t recommend it.

If whole blood can’t be processed right away it’s okay to store it at 4oC for up to two weeks for DNA isolation or for an hour or two for RNA isolation.  Any longer than this and you’ll need to take alternative action.   If you only want DNA you can simply freeze the blood in small aliquots (250-500 μL) at -20oC or -80oC for long term storage.

For RNA extraction from blood, you’ll need to be more vigilant.  RNases are tough enzymes and they can continue to have activity even while frozen.  If the blood cannot be processed quickly then we recommend collecting the blood in an RNA stabilization buffer such as that contained in PAXgene™ Blood RNA Tubes.  In these tubes, intracellular RNA will be stable for three days at 18 to 25°C or five days at 2 to 8°C.

Another option, if you can’t get to your RNA prep right away is to isolate, lyse and freeze the white cell pellet in an RNase inhibiting buffer.   For example, the white pellet can be stored in the PowerMag® WBC Lysis solution contained in the PowerMag Blood DNA/RNA Isolation kit. (See below)  Store the pellets at -20oC or -80oC. Once you are ready to extract, bring the sample to room temperature and proceed with the rest of the protocol.

Getting DNA & RNA from Blood13814735_s

In our PowerMag Blood DNA/RNA Isolation kit, the lysis of RBC and WBC is buffer based and makes use of the fact that in mammalian blood, genomic DNA (gDNA) is only contained in the white cells (leukocytes).  The Red cells (erythrocytes) and platelets lack a nucleus and so neither contains gDNA.  (bird erythrocytes are an exception)   In general, the average number of white cells in 1 mL of human blood is about 7 million.

The first step in our protocol uses a hypotonic lysis buffer that preferentially lyses RBC.  The WBC are pelleted and then the heme containing RBC supernatant is removed.  A chaotrophic buffer is added to the WBC pellet and the nucleic acids are released.  As mentioned above, this is a good point to freeze the sample for future RNA isolation at (-20 C or -80 C) for long term storage if the sample can’t be processed right away.  Our ClearMag® Beads are then used to capture the nucleic acids without binding unwanted contaminants.   Subsequent washes and elution generate ready-to-use DNA and RNA for most any downstream application.

Average RNA and DNA yields?

Not all blood samples are the same. They differ in the number of WBCs and this affects the quantity of nucleic acids isolated from each sample. WBC count can vary based on the health of the subject at the time of blood sampling. Nucleic acid yields vary by species as well.  However, average yields for MO BIO employees were 1 microgram of RNA and 3 micrograms of DNA from 200 microliters of whole (non-vampire) blood.

High Molecular Weight nucleic acids

If you are looking for high molecular weight DNA from blood in a high throughput manner, this kit is the way to go!  We’ve been able to isolate up to 100kb fragments of DNA so if you are looking for targets that are low in abundance or need longer chunks of DNA for sequencing purposes, look to this blood kit!

With all of that said and the vampire in R&D, hunger is looming and its best to get back to the bench where my stock of holy water and garlic bulbs are awaiting my arrival.  Later pumpkin skaters!

References:

  1. Yokota M, Tatsumi N, Nathalang O, Yamada T, Tsuda I. (1999). “Effects of Heparin on Polymerase Chain Reaction for Blood White Cells”. J. Clin. Lab. Anal. 13: 133–140.
  2. DNA isolation by a rapid method from human blood samples: effects of MgCl2, EDTA, storage time, and temperature on DNA yield and quality. Lahiri DK1, Schnabel B. Biochem Genet. 1993 Aug;31(7-8):321-8.

 

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Monster Cryo Tour

Core prep

 

Warning —> If you don’t use twitter, this article might give you the extra boost to go ahead start tweeting away..

I met Arwyn’s tweet one day and rest is history!  He had posted results from his undergraduate class about how there are essential oils such as lavendar that are actually a more effective bacteriocide than other toxic chemicals such as bleach.  Super Rad result!  I got in touch with him and, over time, learned about his incredible and very global climate change relevent research on microbes that live on ice and snow of the Arctic.

I sent him a few basic questions and voilà!  Enjoy.

1. Tell MO BIO about your project

My principal interests are in the interactions between microbes and glacial systems. This summer is very busy for my team as we are working on projects in Svalbard, the Canadian Arctic, Greenland, the Swedish Arctic and the European Alps. At the moment we are wrapping up our Greenland project, which is funded by the Royal Society in the UK. The project aims to build our understanding of how microbial communities on the surface of the Greenland ice sheet change through space and time. Here I have been working with my co-investigators Dr Tristram Irvine-Fynn, also of Aberystwyth University, and Dr. Joseph Cook of Derby University to collect samples and conduct experiments from a science camp on the ice sheet run by the Dark Snow Project.

Midnight Sampling

2. Why is it important?

Not too long ago scientists assumed that glaciers and ice sheets were too hostile for life to survive on them. We know now that microbial life is abundant, active and diverse in form upon, within and beneath glaciers and ice sheets. The microbial population of glacial systems is vast and poorly explored, but microbes play key roles in how glaciers and ice sheets work (DOI: 10.1002/wat2.1029). We recently published some papers on the abundance of microbes at the ice surface (e.g. DOI: 10.1002/cyto.a.22411). To put the numbers into context – well, the media seem to measure glacial disaster in SI units of “Manhattans”. I calculated that an iceberg the size of Manhattan would harbour an equivalent number of microbes in its top few metres of ice as the number of cells in the human population of Manhattan. Just a decade or so ago we had little idea these were habitats for life.
The flipside of this is that glacier melting becomes an ecological problem and not “just” a case of rising sea levels. Microbial habitats on the ice surface are both vulnerable to climate change and amplify its effects. Microbial biofilms and aggregates at the glacier surface, for example cryoconite and ice or snow algae, darken the ice, reducing its albedo and promote melt. In the bluntest of terms the microbes represent a powerful biological feedback to melting at the surface of glaciers and the Greenland ice sheet. We can see “dark zones” of low albedo and intense melting from satellite observations. When you ground-truth these areas you find that these areas are rich in cryoconite ecosystems, and that the “darkness” is derived from humic compounds made by microbes in the cryoconite.

3. What are some potential outcomes?

I hope our project will help map how these microbial communities change over space and time, in particular in relation to melting seasons. Although as I mentioned earlier, we are focused on how microbes cause melting, microbes also respond to melting, and that’s an important facet of our work. Back in 2012, 97% of the surface area of the Greenland ice sheet experienced surface melting, if only for a few days or a week at its extreme limits. On the basis of work on Svalbard glaciers, I hypothesized (doi:10.1038/ismej.2013.51) that some bacterial populations could respond to this melting episode, creating a spatially-expansive but very brief microbial bloom across Greenland in response to the availability of liquid water and nutrients. A massive event in nature but because of its invisible microbial nature, two years later we have no idea if it really happened or not. Last Saturday we lucked out and were able to take samples far inland to see if there are any traces of microbial changes in response to previous melt episodes. I can’t wait to get these samples back to my lab in Aberystwyth.

4. How is your science important to the public?

My team is intensely passionate about how microbes interact with the ice surface. What began as an academic pursuit for us has assumed a broader significance because of the feedbacks between biology and ice melting. We know as the climate warms, Earth’s glaciers and ice sheets will contribute to raising sea levels. This will affect humans across the world, be it people living in low-lying coastal areas which will be at risk of inundation, the hundreds of millions of people that depend on water from sources replenished by glacial melting, or the rest of us that consume food from crops grown in these areas. There is a growing body of evidence that microbial processes considerably accelerate the melting of ice surfaces, so our work plays a small but important part in understanding how ice melt will change our lives.

Core-firn
5. How has MO BIO been helpful?

I’ve been using MO BIO kits for extracting nucleic acid from environmental matrices for over a decade. I must have extracted thousands of samples. In all that time I have only had six or seven samples fail to yield usable DNA! Soils of all kinds, sediments, biofilms, river water, ice melt, snow, air samples. Even cryoconite, which is particularly challenging as while its biomass is relatively low, it is enriched in humic substances that inhibit PCR. MO BIO kits have made environmental genomics much more accessible.

Samples
Secondly, we have increasingly been facing a challenge I refer to as the “rime of the modern glacier biologist” – ice, ice everywhere, and not a lump to freeze! We work in remote environments, far from the nearest ultrafreezer, and liquid nitrogen or dry ice are impractical to use in deep field. Nevertheless, high quality nucleic acids that are representative of the microbial community at the time of sampling are important to us. Stabilizing solutions such as Soil Lifeguard help a lot.

Arwyn, are you kidding me?  Our Planet Earth thanks you,  your science, and your enthusiasm!  Oh, and congrats on your recent Microbiome Awards win, may your rock star science continue!

 

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Working with Salty Soil

Oct 01, 2014
Michelle Tetreault Carlson
Badwater-Storm-1

Badwater Basin Salt Flats, Nevada, USA

Dear MO BIO,

I am using the RNA Power Soil Total RNA Isolation kit with some high saline soil.   I was not able to extract any RNA but was able to extract DNA with the kit.  What’s going on?

Thao

Dear Thao,

Soils or other samples with high salt content can be a little tricky.  The RNA PowerSoil Total RNA Isolation kit makes use of an anion exchange resin to bind and wash nucleic acids.   Under low salt conditions, the negatively charged RNA/DNA are attracted to the positively charged resin and stick.   Under high salt conditions these charges get screened from each other.  By increasing the salt concentration of the buffer on the column, one can control when the RNA and DNA come off the resin.  RNA will come off first and then with increasing salt, DNA will come off.  However, if you start with a high salt content soil you’ll skew the buffer you run over the resin.   This is what happened in your situation.  The residual salt from the soil was high enough to knock off the RNA but not the DNA.

In order to remove the interfering salt from the soil matrix you can wash it.  Add your sample to a sterile 15 ml Collection Tube and then add up to 10 ml of sterile Phosphate Buffered Saline (PBS). Vortex briefly to mix.  Then centrifuge at 10,000 x g for 2 minutes to pellet the soil and microbes.  Remove the liquid.  You may need to do this more than once, repeating up to three times.  However, it seems to work well.

Best,

MO BIO Technical

p.s.

Soils with high levels of metals can also interfere with MO BIO Isolation kits.  For these types of soils we recommend prewashing with a sterile TE buffer (10 mM Tris, 1 mM EDTA).  The EDTA acts as a chelator.   Like above, you can add the TE to a collection tube with your sample and vortex to mix.  Centrifuge to pellet the cells and soil particles and remove the liquid which will contain a higher fraction of metals.  Repeat as needed.

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Wonderful World of Microbes

Sep 08, 2014
Emelia

Enid Gonzalez-OrtaHello!  My name is Enid Gonzalez-Orta and I am an Associate Professor of Biological Sciences at California State University, Sacramento, where I teach undergraduate students about the wonderful world of microbes.  In addition, I embed an authentic research experience into my microbial diversity class each spring where we study soil samples collected from CSU Sacramento Arboretum or from local vernal pool sites.  We study the bacterial community of these samples using traditional methods, like culturing samples onto laboratory media, sequencing of the 16s rRNA gene through the Sanger method, and building phylogenetic trees.  However, I began to think about how much the field of microbial ecology has changed and is changing.  I thought about how culture-independent methods allow us to peek into environments seldom studied in the laboratory and how these methods reveal members of bacterial communities that were previously not known to inhabit these niches.  I also thought about how next generation sequencing (NGS) methods are becoming becoming the “tradition” in this field.  And, I thought about how important it is for undergraduate students to have hands-on experience with bioinformatics and computing in order to interpret the volume of sequencing data that is produced through NGS.  But, how I could I do this when I myself had little-to-no experience with processing 16s rRNA NGS data?  Then came EDAMAME to the rescue!

 

When the opportunity to attend EDAMAME presented itself, I just couldn’t pass it up.  I haven’t been a student for a little while, so I was excited to be in a classroom sitting in a desk and not in the front of the room for a change.  Not only would this course help me teach undergraduate students in my courses how to process NGS data, but it would help to advance my research program CSU Sacramento as well.   My research focuses on the bacterial diversity of the California Vernal Pool Ecosystem in the Sacramento Valley.   This project is done in collaboration with my fellow colleague Dr. Jamie Kneitel and  has been worked on by many undergraduate students.   One of them, Dana Carper, joined my lab as a graduate student focused her Master’s work on this topic .

 

My experience with EDAMAME so far has been awesome.  We are officially half-way through the course and I can say that I have learned a lot from Ashley, Tracy, and Josh.  And, on top of some great instruction, we have amazing guest lectures!  I can’t wait to take what I’ve learned and teach it to my undergraduates.

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