Quote of the Week: Unstoppable Evolution

"Drosophila ... even now is spreading about the world and evolving new species. There is a great irony in the fact that, while the laboratory geneticist is showing his powers over nature by molding his fruit-flies as he pleases, the flies themselves are staging a quiet demonstration of their own."

Harold Oldroyd (1965) The Natural History of Flies. W.W. Norton, New York.

Breaking News

Nature Methods announces the 2008 Method of the Year: Super-Resolution Microscopy.

Web Wednesdays

Today's focus will be on two tech-savvy ways to get answers to your questions about all things Drosophila-related.

A Classic: DroBioNet.

DrosBioNet dates at least as far back as 1993. Based on my experience, topics frequently include job posts, meeting announcements, and requests for advice, protocols and reagents.

Posts are organized by date. Subscribe via email or RSS.

A New Kid: FlyBase Forum.

Another place to view job postings, news and announcements, and ask questions or request protocols--but in an updated on-line environment. Features news and updates from FlyBase. And private messaging among registered users.

Posts are organized by topic. Registration required for many features.

Expect info about forums focused on RNAi in a future post.

Technology Tid-Bits: Very Simple tips for Cost Savings

In a full-genome RNAi screen, in essence thousands of individual experiments are done. Typically, each of these sits in a small well in one of the many 384-well microtiter plates necessary for the full screen.

Why 384-well plate format? One reason is cost savings. 

Working in small volumes (as these plates require), the total amount of reagents and cell culture media needed to do the full-genome screen is much less.  And subsequently, the screen costs less.

Many labs have gotten very good at finding additional routes to cost savings. Stretching grant dollars further and further.  And perhaps with the hard economic times so prominent in the media these days, the issue has been on my mind recently.

So, "use less" is one straightforward way to save costs.  

Another way? Dilute, dilute, dilute.

At the DRSC, we have recently been encouraging researchers to include tests of a minimal effective dilution--particularly of the readout reagents (antibodies, solutions, etc.)--as part of routine optimization for 384-well format.  Even if the minimum you can get away with is a one-to-one dilution, that still means you've cut your costs for that step or reagent in half.

More ideas?  Pipe(tte) up!  Want to hear them.

Hoverfly RNAi

What I'm reading today:

Lemke & Schmidt (2009) Evidence for a composite anterior determinant in the hover fly Episyrphus balteatus (Syrphidae), a cyclorrhaphan fly with an anterodorsal serosa anlage. Development 136, 117-127. [PubMed 19060334]

Why I'm reading it:

I am nothing like an expert in the natural history of flies.  

But I do know this about hoverflies (syrphid flies), they're a popular macro photo pic on Flickr.  They frequent flowers and like bees, do some work as pollinators.

The cover photo on the January 2009 issue of Development shows a hoverfly.  

And the corresponding article includes some RNAi work. In a study of genes that support anterior-posterior patterning (including bicoid and caudal) in a hoverfly, the authors use injection of dsRNA (for RNAi-based knockdown) and mRNA (for ectopic expression) to explore gene function.

It's fun to see RNAi being used successfully in this kind of evolutionary study on a fly that lacks the history of genetics that D. melanogaster has behind it.  

Helps remind me that even though I rarely think about RNAi without high-throughput screens in mind, there are many other ways it's having an impact--on our understanding of Drosophila's many fly cousins and of biology more generally.

Quote of the Week: Fame and Fortune

"Drosophila has now been drawn from its modest retirement in fruit shops and cider mills and has become a public character."

L.C. Dunn (1934) in Heredity and Variation

Protocol Notes

Breaking protocol paper:  

Steinbrink & Boutros (2008) RNAi screening in cultured Drosophila cells. Methods in Molecular Biology. PMID: 18641945.

Also note: Protocols and published protocol papers from the DRSC can be found on our website.

Web Wednesday

The DRSC team recently sifted through a lot of websites to compile a first draft of a links page. 

The idea was to create something helpful for screen hit follow-up. Suggestions for additions are welcome.

Two sites I've stumbled on just this week are DroSpeGe and AAA (for Assembly, Alignment and Annotation; the figure shown here is from their home page). 

Both sites have information about the twelve Drosophila genomes that were published just over a year ago in Nature.

The D. melanogaster genome was published in Science while I was a postdoc (Adams et al. 2000). I still remember the excitement it generated at that year's fly meeting (ADRC). And the scramble to get a copy of the commemorative jigsaw puzzle (or was it a mouse pad?) that was produced to mark the occasion.

At that time, it was possible to compare D. mel sequence with at least partial sequence of the human genome. Sites like Homophila report aspects of those findings. 

Now, we can make different use of the twelve genomes to find sequence elements conserved over much shorter spans of evolutionary time.  

You can BLAST the sequence data here, at FlyBase or at NCBI. And if you're more interested in the actual beasts, you can order them at the fly species stock center, now at UC San Diego.

Direct impact on fly RNAi screening?  

Hard to say. It seems doubtful that we'll have cell lines and dsRNA libraries with which to probe these other species any time soon. And one might be hard pressed to argue that we'd learn much more via that route then we are learning already with RNAi assays in D. mel.

But that's an extremely limited view of the utility of the sequence. The availability of these genomes will certainly inform studies in D. mel--and broader research fields--more generally. And that's a big plus.

Technology Tid-Bits

This week my schedule seems jam-packed with presentations on new technologies and data analysis tools.

It's been fun to see that the number and type of high-throughput image acquisition instruments--as well as the number and type of image analysis software tools--has increased in recent years. No doubt fueled by commercial interest in cell-based assays.

Yesterday, a representative from Definiens came to talk about their latest software for life sciences. It includes analysis of time-course and Z-section image series. 

Seems as if there are many new or improved image analysis tools popping up these days. Not just commercial software. Also things like Cell Profiler that have been developed in academic labs. 

It makes it easy to forget that the early image-based screens at the DRSC were analyzed by that so-expert (but alas, a bit error prone) analysis tool, the human eye + brain.

And today our fellow-HMS pals at the ICCB-L (among other things, they support RNAi screening in mammalian cells) are hosting a representative from Meso Scale Discovery, who will talk about detecting biomarkers using electrochemiluminescence technology.

Electrochemiluminescence? What a mouthful! I'm looking forward to learning more.

What I'm Reading Today

Three recent papers based on screens performed at the DRSC.

Cuttell L, Vaughan A, Silva E, Escaron CJ, Lavine M, Van Goethem E, Eid JP, Quirin M, Franc NC. Undertaker, a Drosophila Junctophilin, links Draper-mediated phagocytosis and calcium homeostasis. Cell. 2008 Oct 31;135(3):524-34.

Zhou R, Hotta I, Denli AM, Hong P, Perrimon N, Hannon GJ. Comparative Analysis of Argonaute-Dependent Small RNA Pathways in Drosophila. Molecular Cell. Volume 32, Issue 4, 21 November 2008, Pages 592-599.

Beller M, Sztalryd C, Southall N, Bell M, Jäckle H, Auld DS, Oliver B. COPI Complex Is a Regulator of Lipid Homeostasis PLoS Biology. Vol. 6, No. 11. *

*Figure 1 from this paper, "Drosophila cells as a Model of Lipid Storage," is shown above (and the figure acts as a link to the paper at PLoS Biology). To view PubMed and other links for all of these citations, visit the DRSC publications page.

Quote of the Week: Man and Fly Walk at the Same Speed (Kind Of)

From On Growth and Form (Sir D'Arcy Thompson):

An apparently simple problem, much less simple than it looks, lies in the act of walking ... The smaller man, or smaller animal, goes slower than the larger, but only in the ratio of the square roots of their linear dimensions ... M. Delisle saw a fly walk 3 in. in half-a-second; this was good steady walking. When we walk 5 miles an hour we go about 99 in. in a second, or 88/6 = 14*7 times the pace of M. Delisle's fly. We should walk at just about the fly's pace if our stature were 1/(14*7) squared, or 1/216 of our present height ... say 76/216 in., or one-third of an inch high. Let us note in passing that the number of legs does not matter, any more than the number of wheels to a coach; the centipede runs none the faster for all his hundred legs.

From an Abridged Edition, J.T. Bonner, editor, Cambridge University Press, 1961, pg. 28-9. On Growth and Form was originally published in 1917.

"Classic" Reads--Edge Effects

What I am reading today:  

A Simple Technique for Reducing Edge Effect in Cell-Based Assays. (2003) Lundholt, Scudder & Pagliaro, Journal of Biomolecular Screening 8(5).  

(Thanks to Amy G. at LiCor for the heads up on the paper.)

Why am I reading it?

Edge effects can be a bane of any plate-based assay. What are they? Let's say you look at your plate-based results and see a pattern that relates not to the biology you want to study but instead, to the position of the wells on the plate. That might be an edge effect. Commonly, the outer wells of the plate will give skewed values and/or contain unhappy cells.

Edge effects have not been a big problem at the DRSC (info on how we accomplish that below). Nonetheless, it's worth thinking about. And exploring ways to keep edge effects at a minimum.

In the paper cited above, the authors describe placing a plate at room temperature prior to incubation at 37 degrees C (their study is on mammalian cells). As fly cells grow at 25 and ambient air is usually about 22, the method might not do much for Drosophila cell-based assays. But if you're visiting us from the mammalian screening world--or interested to do follow-up assays in that system after a fly cell screen--then the quick fix Lundholt et al. propose is probably worth keeping in mind.

How does the DRSC reduce edge effects?  We use a very low-tech tool--wet paper towels--to keep humidity (which appears to be a contributing factor) even and high.  Our incubator is humidified. But nonetheless, our screeners are encouraged to take the extra precaution of putting the plates on top of a layer of damp paper towels and close them up in plastic containers.

This simple solution seems to do the trick.  In general, we see little or no edge effects, and thus our users typically can include the outermost wells in data analysis.

Welcome

Welcome to FlyRNAi, a blog written by the director of the Drosophila RNAi Screening Center (DRSC).  

With information about:

• RNAi and related reagents
• Drosophila cells and assays
• Recent publications
• and more

Please feel free to post questions.

Stephanie Mohr, PhD

DRSC Director