(Press-News.org) Ask any molecular plant biologist about RNA extractions and you might just open up the floodgates to the woes of troubleshooting. RNA extraction is a notoriously tricky and sensitive lab procedure. New protocols out of the University of Florida are quicker, more effective, and more reliable than previous methods.
"Obtaining pure and intact RNA samples is essential for sequencing the active genes, or the transcriptome, of a plant," explains botanist Ingrid Jordon-Thaden, who developed the protocols.
The protocols are given in bench-ready form with detailed notes and a troubleshooting guide in a recent issue of Applications in Plant Sciences. The most successful approach combines the TRIzol reagent and TURBO DNA-free kit manufactured by Ambion, Life Technologies, with additional steps tested by the researchers.
"We needed something that would consistently give us good quality RNA across a wide range of plant types," explains co-author Andre Chanderbali. "Compounds such as flavonoids, tannins, waxes, and other secondary metabolites found in plant tissues can make it difficult to extract RNA."
The researchers were working with a diverse array of woody, aromatic, and aquatic plants while contributing work with a major international transcriptome sequencing project, the 1000 Plants Initiative. They conducted 382 separate RNA extractions to test different techniques on various plant species.
A key ingredient in the new protocols is the same foaming agent that is commonly used in shampoos, sodium lauroyl sarcosinate. Nicknamed sarkosyl, scientists have been using it since the 1970s because it breaks apart cellular fatty membranes to release the contents of a cell.
"Combined with TRIzol and the TURBO kit, sarkosyl helps extract high-quality and -quantity RNA from most plant species," explains Jordon-Thaden. "Adding a CTAB step improved extraction success for even the most stubborn species. Despite the success of the protocols, however, our methods were still not successful for some plants that contain high amounts of mucilage."
In the lab, RNA extractions have a reputation for causing agonizing failed days at the bench. RNA is extremely fragile and sensitive to degradation by enzymes called RNAses that exist everywhere in nature, including the air. Workspaces must be sterilized because RNAses can quickly turn intact RNA strands into a soup of nucleic acids not fit for sequencing.
"As soon as we collect a plant tissue, such as a leaf clipping, for RNA extraction, we flash-freeze it in liquid nitrogen to prevent degradation," says Jordon-Thaden. "We then grind it up and mix it into a cocktail of reagents followed by the washing away of plant fibers and other cellular components. The longer the process takes, the greater the chances of RNA degradation."
"Bad quality RNA equates to bad transcriptome sequencing," says Jordon-Thaden.
Extraction protocols have come a long way since the discovery of nucleic acids, but there is still a long way to go as sequencing projects expand. Commercial kits provide ready-made ingredients and step-by-step instructions that ease the process, but as more labs work with complex non-model plant species, better extraction methods are needed.
The iteration process of troubleshooting 382 extractions and making modifications to manufacturer kits will serve as a valuable resource. As more and more botanists explore the genetic nature of the biodiversity of plants on Earth, these protocols will provide them with an extraction method that can handle almost any plant.
INFORMATION:
Ingrid E. Jordon-Thaden, Andre S. Chanderbali, Matthew A. Gitzendanner, and Douglas E. Soltis. 2015. Modified CTAB and TRIzol protocols improve RNA extraction from chemically complex Embryophyta. Applications in Plant Sciences 3(5): 1400105. doi:10.3732/apps.1400105
Applications in Plant Sciences (APPS) is a monthly, peer-reviewed, open access journal focusing on new tools, technologies, and protocols in all areas of the plant sciences. It is published by the Botanical Society of America, a nonprofit membership society with a mission to promote botany, the field of basic science dealing with the study and inquiry into the form, function, development, diversity, reproduction, evolution, and uses of plants and their interactions within the biosphere. APPS is available as part of BioOne's Open Access collection.
For further information, please contact the APPS staff at apps@botany.org.
Adam C. Levine, M.D., an emergency medicine physician at Rhode Island Hospital found that the World Health Organization's current weight-based guidelines for assessing malnutrition in children with diarrhea are not as reliable as measuring the child's upper arm circumference. His research was published in the Journal of Nutrition.
Diarrhea is common among children who visit health facilities in developing nations. The traditional measures for determining whether a child is moderately or severely malnourished are based on assessing the child's weight directly. Levine found ...
Old-school field work meets cutting-edge technology! For decades, researchers have been making artificial eggs out of plaster, wood, and other materials to test how birds identify and reject the eggs that invading "brood parasites" sometimes sneak into their nests. But these methods have many limitations, and a new study published in the open-access journal PeerJ is the first to test the usefulness of 3D printed eggs for research on egg rejection.
Brood parasites are birds that don't build nests of their own. Instead, they slip their eggs into the nests of other species, ...
Scientists at the Molecular Medicine Institute in Lisbon, Portugal, and at the University College London Ear Institute, United Kingdom, have developed a simple and efficient protocol to generate inner ear hair cells, the cells responsible for our hearing and sense of balance. This study is an important step for the future production of large numbers of these cells for use in cell transplantation therapies or large-scale drug screens. The research has just been published in the scientific journal Development at http://dev.biologists.org/.
Sensory hair cells located in ...
COLUMBUS, Ohio - If you're watching television while using a second screen - like a smartphone or tablet - new research suggests that some of the most expensive marketing messages aimed at you are missing their mark.
While the trend of "second screen" use has become pervasive, this is the first study to show that viewers have trouble recalling brands they see (or hear) on TV if they're using such devices.
"Viewers don't even remember that your brand was there on TV because they were busy posting on Facebook or Twitter or reading email," said Jonathan Jensen, who led ...
Irvine, Calif., May 26, 2015 - The Millennium Drought in southeastern Australia forced Greater Melbourne, a city of 4.3 million people, to successfully implement innovations that hold critical lessons for water-stressed regions around the world, according to findings by UC Irvine and Australian researchers.
It wasn't a new pipeline over the mountains, special rate hikes or a $6 billion desalination plant that kept faucets running. Rather, integrated outreach by utilities and agencies required to work together led to a culture shift among ordinary water users, according ...
CHARLOTTESVILLE, VA (MAY 26, 2015). A surgical skills laboratory and corresponding dissection curricula were established in the Department of Neurosurgery at the Cleveland Clinic in the 2011-2012 academic year. The authors describe how this came about and what it has meant for neurosurgical resident training and assessment of residents' surgical skills in the following paper: "Establishing a surgical skills laboratory and dissection curriculum for neurosurgical residency training" by James K. C. Liu, MD, and colleagues, published today online, ahead of print in the Journal ...
A new study has shed light on how cancer patients' attitudes and beliefs drive the use of complementary and alternative medicine. Published early online in CANCER, a peer-reviewed journal of the American Cancer Society, the findings may help hospitals develop more effective and accessible integrative oncology services for patients.
Although many cancer patients use complementary and alternative medicine, what drives this usage is unclear. To investigate, a team led by Jun Mao, MD and Joshua Bauml, MD, of the Abramson Cancer Center at the University of Pennsylvania's Perelman ...
This news release is available in French.
Hormone therapy (HT) is prescribed to alleviate some of the symptoms of menopause in women. Menopausal women are more likely to be diagnosed with Alzheimer's disease but not other forms of dementia, and HT has been prescribed to treat cognitive decline in post-menopausal women with variable degrees of effectiveness. New research by Dr. Liisa Galea, at the University of British Columbia, suggests the form of estrogens used in HT and previous motherhood could be critical to explain why HT has variable effects. Research in ...
Being very overweight in your teens may double the risk of developing bowel cancer by the time you are middle aged, suggests research published online in the journal Gut.
And a high level of an indicator of systemic inflammation--erythrocyte sedimentation rate, or ESR for short--at this age is also linked to heightened risk of the disease in later life, the study shows.
Adult obesity and inflammation have been associated with an increased risk of bowel cancer, which is the third most common form of cancer among men, worldwide. However, less is known about how obesity ...
Road traffic noise is linked to a heightened risk of developing a mid-riff bulge, indicates research published online in Occupational & Environmental Medicine.
Exposure to a combination of road traffic, rail, and aircraft noise may pose the greatest risk of acquiring a spare tyre--otherwise known as central obesity, and thought to be one of the most harmful types of fat deposition around the body--the findings suggest.
The researchers assessed how much road traffic, rail, and aircraft noise 5075 people living in five suburban and rural areas around Stockholm, Sweden, ...
