RNA sequencing (RNA-seq) is a powerful tool for molecular biologists looking for what drives mechanisms of disease.¹ RNA-seq is routinely used for both research and clinical applications in diseases featuring high levels of genetic dynamism and heterogeneity, such as cancer.² However, although RNA-seq is becoming more accessible and prevalent, it comes with technical obstacles that can prevent scientists from obtaining the high-quality data they need.

RNA is more subject to degradation than DNA, whether chemical, physical, or enzymatic. Complicating the matter, scientists performing disease research rely on clinical tissue samples as RNA sources. These samples are commonly formalin-fixed and paraffin-embedded (FFPE), a process that preserves sample integrity over the long-term for immunohistochemical analysis but can result in highly degraded RNA.²

Scientists are designing FFPE sample-tailored RNA library preparation protocols, reagents, and kits to remove this bottleneck. Twist Bioscience, for example, offers a rapid library preparation kit designed to work with FFPE and other low input samples that takes as little as 4.5 hours to complete from start to finish. The kit protocol includes an optional depletion step for removing ribosomal and globin RNA, leaving only messenger RNA. It also incorporates dUTP nucleotides during second-strand synthesis, allowing scientists to trace amplified sequences back to originating strands.

The Twist RNA Library Preparation Kit is intended to be a strong first step in the RNA-seq process, and therefore is designed to pair with their other target enrichment solutions such as the RNA Exome panel (enriching across the whole transcriptome) or RNA Fusion (targeting specific cancer-relevant fusion genes) panel. Finally, Twist Bioscience can also design custom panels for scientists with a unique application and goal in mind.

Learn more about the available solutions for maximizing RNA sequencing using FFPE samples. 

What RNA sequencing applications do you perform in your research?

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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) uses an electric current to separate proteins in a heterogeneous sample according to molecular weight and is one of the most commonly used techniques in the life sciences.^1,2 The polyacrylamide gel is an integral part of SDS-PAGE. However, despite the technique being over 50 years old, most researchers still cast gels by hand via a time-consuming process that can take 90 minutes or longer. Furthermore, scientists create these gels using the toxic compounds ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) to catalyze the polymerization of acrylamide and bisacrylamide into polyacrylamide.

         

Pre-cast gels are commercially available, but they are expensive and their packaging tends to generate considerably more waste than hand-casting. Moreover, researchers cannot modify the composition of pre-cast gels to fit their experiments.² All of this ultimately creates a dilemma where scientists must choose between time, resources, and flexibility. As such, pre-cast gels have not superseded hand-casting as the preferred method among the scientific community.

Clearly, new innovations are needed for this old workhorse technique, and the mPAGE^® Lux Casting System provides a novel solution. Designed to provide flexibility and reliability without incurring costs in terms of time, resources, or waste, the mPAGE^® Lux Casting System uses three pre-made reagents and a curing station to produce gels in a process that takes about three minutes. The system generates Bis-Tris gels as opposed to commonly used Tris-Glycine gels, enabling shorter run times and longer shelf lives. The system further removed the need for APS and TEMED, which are toxic compounds. Finally, because gels can be rapidly made on-demand, researchers no longer have to risk wasting resources on pre-preparing gels that will go unused.

Learn more about how a new system lets researchers produce SDS-PAGE gels on-demand and to their own specifications in three minutes. 

What is your biggest western blotting pet peeve?

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Evgeny Kvon, a geneticist at the University of California, Irvine, fell in love with molecular biology when he learned about the experiments that led to the discovery of DNA as the carrier of genetic information in high school. Now, as a group leader, Kvon explores how enhancers, which are non-coding regulatory DNA elements, orchestrate the precise spatiotemporal transcription of genes during mammalian development.

How does your team study enhancer function?

We use transgenic mouse embryos to show that a DNA sequence is an enhancer. In one assay, we clone the enhancer sequence upstream of a promoter and lacZ reporter gene and then inject this construct into the mouse egg. By looking at lacZ activity, which appears as a blue color in the mouse embryo, we can track the location and activity of that potential enhancer. We also use high-throughput chromosome conformation capture techniques to study enhancers and their chromatin interactions. Using both methods, we recently mapped the enhancer-promoter interactions of almost 1000 known enhancers in different mouse embryo tissues and found that around 60 percent of them skip the promoters of neighboring genes and contact more distant genes.¹

What are some of the unanswered questions that you would like to explore in the coming years?

How enhancers know which genes to ignore and which to activate is an interesting question that needs further investigation. It is also unclear how remote enhancers can act on their target genes even though they are located far away from one another. We started to tackle this question in a recent preprint, and we found that a long-range enhancer has an element, which we named range extender (REX), right next to it.² When we added REX to a short-range enhancer, it gained the ability to act long range. We believe we identified a genetic signature that allows remote enhancers to work that way.     

This interview has been edited for length and clarity.