Webinar: The How’s and Why’s of the Early Steps in RNA Analysis

Ribbon diagram of RNA’s biggest threat: a ribonuclease

Ribbon diagram of RNA’s biggest threat: a ribonuclease

RNA analysis from RT-pPCR to RNA-seq has become an increasingly important part of life science research as we seek to understand gene expression patterns, cell signaling and developmental events. To be successful at these RNA analysis steps, however, the upstream RNA purification needs to produce intact, high-quality product suitable for downstream work. Many RNA purification systems are available, ranging from high-throughput to manual using a variety of chemistries. You can purify RNA from FFPE or fresh mammalian tissues. How do you know which system to choose and when to use it? Our free webinar on August 11, The Hows and Whys of Early Steps in RNA Analysis, describes different methods for purifying RNA from fresh or fixed samples, protecting it from degradation and assessing quality before you proceed with downstream work. Register today to learn how you can achieve the best results possible with your RNA analysis studies.

Other Resources

DNA and RNA Purification Product Selector

Working with RNA (blog)

About the Webinar Series

www.promega.com/webinars/ provides a schedule of upcoming webinars. In addition there are links to previous webinars that allow you to either view the recording or download a pdf of the presentation. There is also a pdf of additional material available for each past webinar.

To register for a webinar, use the “registration” link at: www.promega.com/webinars/ This allows you to view the webinar and participate in the live chat. Need a reminder? You can also sign-up for monthly invitations to webinars at the webinars page. Note: Live chat is only available for live webinars, not links to recorded webinars.

Summer Friday Blog: The Tallgrass Prairie in the American Midwest

The tallgrass prairies of the great plains in the United States are in their summer glory right now, so I thought we would take a walk through the prairie and share some pictures. Eight foot tall compass plants are painting their yellow glory against the deep greens of late summer and the contrasting clear blue skies. Butterflies of all types–monarchs, viceroys, red admirals and others are visiting the showy flowers and munching on the milkweeds. Sandhill cranes are teaching their young to fly. Dragonflies are looping and buzzing in the skies above.

Daisies and other flora where the prairie meets the woods. Madison, WI. photocredit: Michele Arduengo

Daisies and other flora where the prairie meets the woods. Madison, WI. photocredit: Michele Arduengo

Continue reading

All You Need is Pla (for Pneumonic Plague)

Yersinia pestis. By Mrs Robinson at bg.wikipedia (Transferred from bg.wikipedia) [Public domain], from Wikimedia Commons.

Writing about Yersinia pestis or the Black Death, has earned me a reputation among Promega Connections bloggers. I am interested in what researchers have been able to piece together about the causative agent of ancient plagues, what modern research shows about how Y. pestis spreads in the body and the continuing reservoirs in modern times, resulting in publication of eight blog posts on the subject. Understanding Y. pestis bacterium is of continuing interest to researchers. How did Yersinia pestis evolve from the humble Yersinia pseudotuberculosis, a pathogen that causes gastrointestinal distress, into a virulent pneumonic plague that is a global killer? One strategy for answering this question is to look at the genomic tree of Y. pestis and trace which strains had what characteristics. In a recent Nature Communications article, Zimbler et al. explored the role of the plasmid pPCP1 in Y. pestis evolution and the signature protease Pla it expresses. Continue reading

Ancient DNA: Futuristic Technology Brings the Past into the Present

1781140_lIsolating and sequencing DNA from ancient samples is a highly specialized field of research that easily captures the imagination. For me it started in the early 90’s when I read about researchers using PCR (a relatively new technique at the time) to amplify, and subsequently sequence, the mitochondrial DNA of an extinct subspecies of zebra using a sample collected from a skin rug found at an estate in England.

From samples a few hundred years old to ones that are thousands of years old, scientists have made good use of technological advances to push back the boundaries of time. In this video from Science, Evolutionary Biologist Beth Shapiro talks about working with ancient DNA, and how new advances such as Next Generation Sequencing have made it possible to gather more information from ancient samples.

Science put together a Special Issue focused entirely on the research surrounding ancient DNA. You can find all the articles in this Special Issue here:

Special Issue: From mammoths to Neandertals, ancient DNA unlocks the mysteries of the past.

 

Summer Friday Blog: Journey into Outer Space for the Delta Aquarid Meteor Shower

11156716_lThis week we travel to outer space, the Final Frontier, to catch a glimpse of the Southern Delta Aquarid Meteor Shower. But don’t worry, you don’t have to leave your backyard. Just grab a blanket and find a place without too much light pollution, and you’ll be able to catch a glimpse of this worldwide phenomenon.

The Southern Delta Aquarid Meteor Shower is an annual summer occurrence that spans July and August, but this weekend marks its nominal peak in activity. From July 26th through August 1st, give or take a couple of days because nature is lovably fickle, we can expect a maximum hourly rate of 15-20 meteors. That might not sound like much, but a special angle of atmospheric entry gives Delta Aquarid meteors long, lingering trails that seriously set this shower apart.

Most meteor showers are created by comets. As a comet circles our Sun, it sheds a rocky dust stream along its orbit. When Earth travels through this space litter, the result is a meteor shower. Astronomers believe that the Southern Delta Aquarids originated from the breakup of two sungrazing comets, Marsden and Kracht.

Shooting stars, as they’re lovingly called, can appear anywhere in the sky, but if you trace the tails it becomes clear that each shower has a definite epicenter. The showers are named after these radiant points, taking the name of the constellation dominating that particular region of the sky. The Delta Aquarids, as you can probably guess, pay homage to the constellation Aquarius. Look for the star Skat within the “water bearer” constellation, the point where Delta Aquari meteors are born.

For every time zone and all continents, the hours between midnight and dawn will be the best time to glimpse these brief celestial bodies. Those of you in the southern hemisphere and southerly latitudes in the northern hemisphere will get a better show, as is typical with this particular shower, though all observers are bound to see activity. Unfortunately, this year the waning crescent moon rises around midnight and will drown out dimmer meteors. But we’ll still see the big ones, and those are arguably the most thrilling.

Meteors are really just bits of interplanetary debris traveling tens of thousands of miles per hour, igniting as they vaporize in Earth’s upper atmosphere, but they sure are pretty. For a preview, take a look at this video of last year’s meteor shower, filmed August 2 by Canadian Geographic.

Sample preparation: A critical step for consistent protein phosphorylation data

MSextractcroppedProtein phosphorylation is a very important protein post-translational modification that controls many cellular processes including metabolism, transcriptional and translation regulation, degradation of proteins, cellular signaling and communication, proliferation, differentiation, and cell survival (1). Approximately 35% of human proteins are phosphorylated. Phosphoproteins are low in abundance, and, therefore, are challenging to detect and characterize by mass spectrometry. Different enrichment systems have been developed to isolate phosphopeptides. Among these techniques, immobilized metal affinity chromatography (IMAC) using Fe3+ and Ga3+ has been widely used for the enrichment of phosphopeptides.

Typical experimental workflows are tedious and consist of numerous steps, including sample collection and cell lysis. One of the major challenges of the process is to maintain the in vivo phosphorylation state of the proteins throughout the preparation process

To evaluate the effect of sample collection protocols on the global phosphorylation status of the cell, a recent paper by Kashin et al. compared different sample workflows by metabolic labeling and quantitative mass spectrometry on Saccharomyces cerevisiae cell cultures (2).

Three different sample collection workflows were evaluated: two that used denaturating conditions and involved mixing of cell cultures with an excess of either ethanol (EtOH) at −80 °C or trichloroacetic acid (TCA), and a third under nondenaturing conditions and washing the cells in PBS.

Their data suggest that either TCA or EtOH sample collection protocols introduced lower collection bias than the PBS protocol. It was also suggested that similar studies be carried out to determine what effects sample preparation has on other post translation modifications such as acetylation or ubiquitination.

Literature Cited

  1. Thingholm T.E. et al, (2009) Analytical strategies for phosphoproteomics. Proteomics 9,1451–68
  2. Kanshin, E. et al. (2015)  Sample Collection Method Bias Effects in Quantitative Phosphoproteomics. J  Proteome Res. 14, 2998-04.

A Reason for Ribonuclease: From Laboratory Nuisance to Cancer Therapeutic

"RNase A". Licensed under CC BY-SA 2.5 via Wikimedia Commons - https://commons.wikimedia.org/wiki/File:RNase_A.png#/media/File:RNase_A.png

“RNase A”. Licensed under CC BY-SA 2.5 via Wikimedia Commons – https://commons.wikimedia.org/wiki/File:RNase_A.png#/media/File:RNase_A.png

RNase, back in the early 1990s, posed a serious threat to laboratories working with RNA isolation. My graduate work involved isolating RNA from the tissues of Lyme disease-infected mice and hamsters. We struggled to DEPC-treat glass and plasticware, or autoclave anything that could be autoclaved, kept tissues cold during RNA harvest and held our breaths (truly, as aerosol could be another source of ribonuclease) until PAGE proved us successful in RNA isolation.

Ribonuclease (RNase) was omnipresent and the arch rival of our work, across several species, due to its RNA destroying abilities.

Now, a July 13, 2015 publication by researchers at the University of Wisconsin-Madison provided both a catch-up for this former lab rat on modern day research with and knowledge of RNase, as well as an exciting look at what may be a real purpose for this RNA-destroying molecule: RNase has moved to clinical trials due to the discovery of it’s cytotoxicity for cancer cells.

Raines’ group in the Department of Chemistry at UWI-Madison published in ACS Central Science their findings on the ligand that RNase 1 uses to attach to human cancer cells, in the article, “Human Cancer Antigen Globo H is a Cell-Surface Ligand for Human Ribonuclease 1”. Continue reading

Think your budget is too small to start your new lab? Well, think again.


eh62Guest blog by Joanna Stevenson, Promega AG

You are young, dynamic and full of initiative. Your dreams oscillate around setting your playground and working without being told what to do and how to do it. You think it is the only way for you to produce results that others will envy. Well, your dreams can actually come true sooner than you think— with your persistence and with a little help from the Promega New Lab Set Up Program.

Even after receiving for external funding, you probably have a few items you still need to set up your new lab. Maybe you have already established your first lab, but you need to change the location. In any case, maximize you budget and continue dreaming. It doesn’t matter if you are in the USA, Australia, Spain or Switzerland –we can help. Please visit our program at www.promega.com/newlab to find out how.

Summer Friday Blog: This Week We Travel to Hawaii and Peshtigo, WI to Learn about Firestorms

Fire-whirlThis week’s video takes us to a forest fire on Mauna Kea, a dormant volcano on the island of Hawaii. One of the firefighters captured this amazing video of a fire whirl that erupted as the air temperature near the ground grew very hot. Fire whirls like this one are caused by extreme heat rising from the ground rather than a confluence of atmospheric events, but they can be be every bit as destructive as atmospheric tornadoes and cause a forest fire to continue to burn out of control.

There are written records of fire tornadoes including several that developed after lightning struck an oil storage facility near San Luis Obispo, CA, USA in 1926. In 2003, scientists confirmed true fire tornadoes in Australia associated with the Canberra fires. In this case the fire tornadoes produced damage consistent with the intensity of an F2 tornado. In The Great Pestigo Fire in 1871, the town of Peshtigo, WI, may well have been consumed by fire tornadoes. Dry weather conditions and slash and burn farming practices contributed to this devastating fire (as they did the more famous Chicago fire that occurred on the same day). Strong winds carried a forest fire into the mill town of Peshtigo, WI, and researchers theorize that cut timber and wooden structures of the town fueled such intense heat that a massive fire whirls formed, consuming the town. You can read some compelling stories about the Peshtigo fire here and here.

Understanding the conditions under which firestorms and fire tornadoes form hopefully will lead to a better understanding of how forest and brush fires spread and allow scientists and fire control experts to develop more effective methods of control.