Riboprobes are RNA probes that can be produced by in vitro transcription of cloned DNA inserted in a suitable plasmid downstream of a viral promoter.
Viruses code for their own RNA polymerases, which are highly specific for the viral promoters. Using these enzymes, labeled NTPs, and inserts in both forward and reverse orientations, both sense and antisense riboprobes can be generated from a cloned gene.
Transcription of RNA is performed with the appropriate RNA polymerase (T3, T7 or SP6), depending on the RNA polymerase promoter sites present in the chosen vector. Because these polymerases are extremely promoter-specific (i.e., there is almost no transcriptional cross talk), virtually homogeneous RNA can be obtained using plasmid DNA as the template in a transcription reaction. When it is desirable to copy only insert DNA sequences, the plasmid is linearized at an appropriate restriction site before the transcription reaction and only discrete “run-off” transcripts are obtained, virtually free of vector sequences. RNA transcripts may be used to generate radioactive probes for hybridization to Northern and Southern blots, plaque and colony lifts as well as non-radioactive probes (i.e, labeled with digoxgenin)for in situ hybridization.
Recent references using riboprobes include: Continue reading
Differential contrast image of HCT116 colon cancer spheroid grown in a 96-well hanging-drop platform after seeding with 800 cells. Copyright Promega Corporation.
Tissue culture using primary or cultured cell lines has long been a mainstay of testing compounds for inhibiting cell growth or promoting apoptosis during screening for cancer drugs. However, the standard culture conditions result in monolayers of cells, dividing and growing across the bottom of a well, plate or flask in a single layer. The drawback of this technique is that organisms do not come in monolayers; a three-dimensional (3D) spheroid is closer to the in vivo state, especially if the spheroids are made up of more than one cell type like tumors in multicellular organisms. Even more beneficial would be using 3D cultured cells in high-throughput screening to facilitate compound profiling for target effectiveness and cytotoxicity. In a recent PLOS ONE
article, researchers used normal and breast cancer cells both in monoculture and coculture to test a set of compounds and found results differed between 2D and 3D cultured cells. Continue reading
This Promega Notes Cover from 2004 celebrated the potential stop and go power of DNA-directed RNAi.
In the early 2000s, RNAi was a hot topic. The science world was abuzz with all the possibilities that harnessing this natural process could hold. And why not? The idea of posttranscriptionally silencing genes using only a small fragment of double-stranded RNA is huge—big enough to earn the scientists who discovered it a Nobel Prize in 2006.
The process of RNAi starts with short (~70 nucleotieds), double-stranded fragments of RNA called short hairpin RNAs (shRNA). These shRNAs are exported into the cytoplasm and cleaved by the enzyme Dicer into smaller pieces of RNA that are about 21 nucleotides long and are referred to as small interfering RNAs (siRNA). The siRNAs reduce or stop expression of proteins through a sequence of events where the antisense strand of the siRNA is incorporated into and RNA-induced silencing complex (RISC), which then attaches to and degrades its complimentary messenger RNA, thereby reducing or completely stopping expression.
It turned out, however, that harnessing the promise of RNAi was a little trickier than anticipated. Continue reading
Not a wolf, this dog is an Australian shepherd.
Research from the American Behavior Society was noted this week in Science (22-August-2014). The title, “Wolves Cooperate but Dogs Submit, Study Suggests” caught my attention, perhaps because I was at work doing my job as a science writer/editor and the word “dog” appeared in something related to work. What could be more fun than dogs at work?
But my attention was sustained because this is yet another report comparing wolves and dogs, a natural and obvious comparison, but one that always puzzles me, for several reasons.
As a former graduate student and lab tech, I know that when doing research, results are everything. Wait: Correct interpretation and reporting of results is the ultimate. But without the proper controls, one cannot correctly interpret or report results.
The control is the piece or part of the experiment that shows what happens when no treatment is applied; the sample or subject is in the same environment and has the same experience as the treated samples or group.
However, my experience is in biology research. The dog-wolf study discussed here is behavorial research. I have never (willingly and knowingly) participated in behavioral research.
But I know that controls are essential and that in behavioral research on live subjects, controls are probably very difficult to…control. Continue reading
GloMax® Discover Multimode Reader with injectors.
Luciferase assays are useful tools for studying a wide range of biological questions. They can be performed easily by adding a reagent that provides components necessary to generate a luminescent signal directly to cells or a cell lysate. However, once this reagent has been added, how long you wait to measure the signal becomes a key consideration in generating consistent data. Dependent on which luciferase assay you use, you may need a luminometer that can use injectors to deliver the assay reagents. The reason for this is simple, but can be confusing to new users.
Let’s start by discussing two types of luciferase assays: “flash” vs. “glow”. Continue reading
A wanted poster for Jack the Ripper, who was also known as Leather Apron.
Image courtesy of the British Museum
In the late 1800s, Victorian England was mesmerized and horrified by a series of brutal killings in the crowded and impoverished Whitechapel district. The serial killer, who became known as “Jack the Ripper
”, had murdered and mutilated at least five women, many of whom worked as prostitutes in the slums around London. None of these murders were ever solved, and Jack the Ripper was never identified, although investigators interviewed more than 2,000 people and named more than 100 suspects
. Now, 126 years after the murders, a British author, who coincidentally has just published a book on the subject, is claiming that DNA analysis has revealed the identity of the notorious killer. DNA is often thought to be the “gold standard” of human identification techniques, so why is there so much skepticism surrounding this identification?
FIGURE 1: Foldscope design, components and usage.
(A) CAD layout of Foldscope paper components on an A4 sheet. (B) Schematic of an assembled Foldscope illustrating panning, and (C) cross-sectional view illustrating flexure-based focusing. (D) Foldscope components and tools used in the assembly, including Foldscope paper components, ball lens, button-cell battery, surface-mounted LED, switch, copper tape and polymeric filters. (E) Different modalities assembled from colored paper stock. (F) Novice users demonstrating the technique for using the Foldscope. (G) Demonstration of the field-rugged design, such as stomping under foot.
Scientific inquiry —looking at the world and asking questions about what we observe—is a natural human behavior. Why is the sky blue? What would happen if I did this Mom? Ask any grade school teacher. Kids do science naturally. They are not afraid of questions. They are not afraid of nature. They are not afraid of experiments and data collection.
One other things kids do really well is: fold paper. I never cease to be amazed at the elaborate fortune tellers, hoppers, boats, hats and other creations that my daughter and her friends make at a moment’s notice out of virtually any scrap of paper they can find.
Recently members of the Prakash Lab at Standford University announced the Foldscope: an optical microscope that is printed and folded from a single flat sheet of paper. These microscopes, which can provide magnification of up to 2000X, can be produced for less than $1.00/each. Furthermore these scopes weigh less than 10g (a couple of coins), require no external power source, can be dropped from 3-stories without damage, and can even be stepped on.
These characteristics make the Foldscope ideal for field work, particularly in remote locations where access to power and other resources is difficult. Prakash and colleagues have published their work in a PLOS One paper and have demonstrated many uses for these Foldscopes including high-resolution brightfield microscopy, fluorescence microscopy, and darkfield microscopy. Continue reading
CellTox™ Green Dye Binds DNA Released By Dying Cells
If you have ever wondered about the differences between the various cell viability and cytotoxicity assays available, why you would choose one over another or how they can be used together, tune in to the webinar “A Real-Time Cytotoxicity Assay That Delivers More Relevant Data” (Tuesday, Sept 10). In this webinar, Promega Scientist Drew Niles explains how various metabolism and biomarker-based viability and cytotoxicity assays work, and describes how they can be used most effectively to give maximum information about mechanism and timing of cell death.
“Is a cell treatment toxic?” and “Why are the cells dying?” are questions that can be difficult to answer simply. The answer depends on dosage, treatment time, mechanism of action of the test compound, and the cell type used—and may sometimes be limited by features of the assay itself. For example, many viability and cytotoxicity assays measure biomarkers that are themselves subject to degradation over the course of longer experiments, complicating the interpretation of results. Drew provides an explanation of these issues and illustrates the critical role of timing in deciding on the assay to use and in interpreting results. Continue reading
As we start a new year in our labs, we thought this cartoon reminding us of some lab etiquette might be useful. Enjoy!
Group Photo from the 2014 Core Techniques in Protein and Genetic Engineering Course Held at the BTC Institute July 14-18. Photo credit: BTCI
One of the things that I encourage all of the students I interact with in BTC Institute courses to do in order to boost retention and make meaning out of the activities that we do in class is to reflect on their experiences. Reflection is one way to connect new knowledge to past experience and get it to really stick in the brain, among other things.
Taking my own advice to heart, I use this space to ponder some interesting aspects of these experiences from my own perspective. This summer, I worked with 65 students and over 25 instructors to deliver four weeks of intensive instruction in molecular biology applied to a wide range of research areas. Continue reading