The BiosearchTech Blog

To truly understand complex diseases such as cancer, scientists need access to tools that don’t just average data across millions of cells, but that can interrogate single cells to examine  gene expression and regulation in diseased cells. 

 It wouldn't be fair to close out 2016 without showcasing the stellar year that Stellaris^® RNA FISH has had. Take a closer look at our top 10 highlights from the past year - mind blowing images are only the beginning!

Here is the final installment of our “Imaging Stellaris Assays” blog series. In our two previous posts, we’ve gone over microscope hardware and image acquisition.  In this article, we will walk you through some simple downstream image processing that will turn your raw Z-stack images into spectacular-looking, publication-ready images.

We have recently released a Stellaris^® RNA FISH Citation Center to make it easy to find publications related to your areas of interest. Incredibly, a full third of these hundreds of papers are published in Nature, Science, or Cell, indicating both the tremendously high quality of our customer’s work as well as the potential value of including Stellaris RNA FISH in your publications. The number of citing publications is growing precipitously each year and now includes a wealth of protocols, references, and technologies including super-resolution and STORM microscopy, whole mount, targets including viruses and plants, CLARITY, and other cutting edge applications. The citation center is updated several times a month to ensure that the most recent publications are always available.

Stellaris® RNA FISH is a novel technology that allows for the detection of single or clustered RNAs in fixed cells and tissue. Multiple unique probes with similar stability are designed to hybridize along the length of a single RNA target and upon hybridization, generate a fluorescent signal. LGC Biosearch Technologies offers customers a web-based, free, and easy to use software for Stellaris RNA FISH Probe design. The probe designer takes an input sequence, such as an mRNA coding sequence, and outputs a set of up to 48 probes designed for optimal binding properties.

Here we continue our “Imaging Stellaris Assays” blog series.  We last left off talking about the most important components: the microscope and the hardware features necessary to detect your Stellaris signal. In this blog article, we will walk you through the technique of finding your cells and acquiring a sharp and stunning image. 

The cleavage and polyadenylation event, the final trimming in the molecular mRNA barbershop, normally releases both the finished styled mRNA, and the stylist. But, in salt-stressed cells, as there are fewer transcription starts, the polymerase stays busy, and keeps on going, and going, down the template strand as if there were no end in sight. Then, as the cell has finished acclimatizing to the new environment, and as promoter-guided gene transcription starts up again, the previously unwanted RNA growth can now serve as a guide for the barber to find a chair with a new customer in need of styling. RNA-style. Cap first, then splice, and lastly, trim the ends.

Stellaris® FISH is an RNA detection technology performed by means of in situ hybridization. This technology and all of the incredible applications it supports would not exist without a detection tool. The detector in this scenario is a fluorescence microscope. 

The microscope is the single major limitation for scientists who are new to the Stellaris technology. The reason being – well, this is a new technology and fluorescence microscopes are not ordinary instruments found in every lab across the country. These are highly intricate, sensitive, and costly pieces of real estate that reside primarily in core laboratories to provide additional interrogation of biological systems through optics. With that said, every microscope system is unique. In other words, it’s not a packaged deal, but rather entirely customized. From the nuts and bolts of the hardware to the software that coordinates all your movements, every piece is different from one lab to the next. Streamlining a protocol for each and every combination of system imaginable would be impractical. So whether your lab uses Nikon, Leica, Olympus, Zeiss, or something else, this blog article is intended to share fundamental microscope requirements to help you obtain a great Stellaris image.

Studying the expression level of RNA transcripts is often complicated by the fact that many DNA sequences are transcribed into multiple RNA isoforms. Or, in cases with microbes, each strain can represent a different sequence variant due to the frequency of mutations in these organisms. Despite these complications, it is still possible (in some, but not all, cases) to design a functional assay to detect multiple variants or isoforms of a gene or transcript. In other words, design one assay to specifically detect multiple targets. In this article, we will go over the basics of designing your assay to detect multiple transcripts at once.

Gene expression and molecular biology research in vision and ophthalmology presents unique challenges to researchers and clinicians due to the complexity of the eye. The eye consists of many structures containing specific cell populations, each exhibiting different expression profiles. Such an intricate organ requires a series of advanced methods in your molecular toolkit to unlock the mechanisms of degenerative eye disease.

This past April, Biosearch attended the American Association of Cancer Research (AACR) conference in Philadelphia, PA where, in collaboration with Byung Chul Kim from Clinomics, Inc, we shed light on the detection of fusion genes by means of Stellaris® RNA FISH. Gene fusions have the potential to activate otherwise silent signaling enzymes, such as the anaplastic lymphoma receptor tyrosine kinase (ALK), leading to significant numbers of lung and other related cancers. This particular gene fusion occurs from inversions on chromosome 2 (inv2(p21p23)), fusing the EML4 and ALK genes and connecting the promoter and 5' half of EML4 (active in normal lung tissue) together with the 3' and kinase encoding half of the otherwise silent ALK gene.

A recent paper published in Molecular Cell by Dimitrova et al. from the lab of Dr. Tyler Jacks at MIT, used Stellaris® RNA FISH to help answer questions regarding whether a pro-apoptotic lncRNA (lncRNA-p21) can regulate nearby genes via cis- or trans-acting mechanisms¹. Long non-coding RNA-p21 and p21 expression is stimulated in response to the activation of the p53 pathway. This pathway is an important area of study because it is involved in tumor suppression and is elicited in response to DNA damage and cellular stress.

Buffers are often overlooked and taken for granted until the day comes when a peculiar result is observed and its origin is traced to a bad buffer. Although in very rare cases mistakes in the composition of buffers have led to discoveries such as the correct number of human chromosomes¹, using properly prepared buffers can be the key to success with technologies such as Stellaris RNA FISH.

Neuroscience gene expression research can come with many hurdles, especially when working with brain tissue or mixed populations of cultured neurons. Here are just a few obstacles Neuroscientists run into:

1. The brain is not made only of neurons! Alongside neurons, brain tissue can contain a variety of cell types such as glial cells and oligodendrocytes.These cell types may act separately or in conjunction with each other to perform a specific function.
2. The brain itself is regionally heterogeneous, containing specialized anatomical structures such as the amygdala, hypothalamus, and hippocampus. These regions perform distinct functions such as eliciting the fight or flight response, signaling reproduction, or contributing to memory.
3. Even so, delving within each of these brain regions, you will find even more specialization. Certain cell populations express a particular RNA signature. For example, tyrosine hydroxylase (Th) expression in dopaminergic neurons, which is critical to the synthesis of the neurotransmitter dopamine. Cells surrounding Th cells, or interspersed with Th cells do not express Th at all! These are all contributing factors that make neuroscience so incredibly fascinating, yet so cumbersome.

Stellaris controls, who needs ‘em? We all do. Especially if it’s your first time interrogating a sample with a new Stellaris RNA FISH probe set, it is critical to consider the proper controls. The concept of positive and negative controls for experiments is not a new one. Far too often, we tend to bypass the consideration of controls in favor of saving time. However, it really all boils down to, “how do you, as a scientist, interpret your results after the experiment is over?”

Positive and negative controls for Stellaris experiments can be viewed from multiple angles. Let’s say you were successful in measuring spots on your first try, the major questions you may ask are:

• Is the signal I am measuring being produced by probe bound to RNA?
• Are these spots true signal or just background?
• Are these spots specific to my target or are they picking up other RNAs?