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.
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.
Formalin-fixed, paraffin-embedded (FFPE) tissue samples are extremely common sample types. In this form, tissue is easy to store for extremely long periods of time and useful for immunohistochemical studies. Additionally FFPE samples are fairly inexpensive to produce. However the formalin fixation procedure, which was developed long before the advent of molecular biology, results in chemical crosslinking of nucleic acid and protein molecules inside the cells. This crosslinking presents a challenge for isolating intact, high-quality nucleic acid DNA; so getting at the wealth of molecular information within an FFPE sample can be difficult.
The polymerase chain reaction (PCR) has revolutionized modern biology as a quick and easy way to generate amazing amounts of genomic data. However, when PCR doesn’t work, it can be frustrating. At these times, PCR and reverse transcription PCR (RT-PCR) inhibitors seem to be everywhere: They lie dormant in your starting material and can co-purify with the template of interest, and they can be introduced during sample handling or reaction setup. The effects of these inhibitors can range from partial inhibition and underestimation of the target nucleic acid amount to complete amplification failure. What is a scientist to do?
microRNAs (miRNA) are abundant RNA molecules around 21 nucleotides long that regulate specific mRNA expression by directly interacting with the mRNA molecule. Our understanding of miRNA function in mRNA regulation has grown exponentially as more miRNA molecules have been described. As of 2013, more than 24,000 miRNA molecules had been described from more than 140 separate species, indicating that miRNA regulation is conserved across species. In humans, 2,500 mature miRNAs have been described, and researchers predict that 60% of human protein-coding genes may be targets of miRNA regulation. Most often miRNA regulation of an mRNA results in decreased expression, either by destabilizing the mRNA or by inducing translational repression. Very recently, some researchers have reported up regulation of mRNA through miRNA activity.
Since miRNA molecules are so abundant within cells and across species and their target sequences are found in so many protein-coding genes, understanding how miRNA regulation of mRNAs acts in concert with the many other levels of gene expression regulation becomes a complex, but fundamental, biological question.
In-gel digestion complete in only 1 hour.[/caption]Identification of proteins resolved by SDS-PAGE requires a lengthy in-gel digestion and extraction procedure. The publication, Mass Spectrometry Compatible Surfactant for the Optimized In-Gel Protein Digestion, Saveliev, S. et al. (2013) Anal. Chem.85, 907–14, addressed these obstacles by illustrating the technical benefits of the ProteaseMAX™ mass spectrometry surfactant. A recent webinar reviewed key aspects of that paper that included:
Improved peptide recovery from gels: ProteaseMAX™ improved identification of proteins by enhanced protein digestion, increased peptide extraction and minimized postdigestion peptide loss. This is a major benefit for the in-gel digestion of low abundant proteins and enables the use of minimal sample material.
The webinar also contained information regarding the how the structure of ProteaseMAX™ Surfactant enables it to compatible with mass spec and how it can be used for protein denaturation and solubilization.
About the Webinar Series
www.promega.com/webinars/ provides a schedule of upcoming webinars. In addition, there are links to previous webinars, which allow you to 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 a live webinar and participate in the live chat.
Need a reminder? You can also sign-up for monthly invitations to webinars at the webinars page (see link above). Note: Live chat is only available for live webinars, not recorded webinars.
It’s a scientist’s nightmare: Spending time and resources to investigate a biological phenomenon only to learn later that your cells are not what you think they are—their true identities hidden. As a result, all of the data that you’ve generated with those cells, published and unpublished, are cast into doubt. You thought that you knew your cells, that you could trust them, but your trust was misplaced. At some point, perhaps even before the traitorous cell line entered your laboratory, the cells were mislabeled, misidentified or contaminated with another cell line. It didn’t have to be this way. There are easy steps you can take to prevent the headache and heartache of cell line misidentification and contamination.
Today we can see inside the cell and identify protein interactions in their native environment. Many proteins have been characterized in a macromolecular complex, in an individual cell, or in the whole organism. We study proteins in their native environment because they rarely work in isolation. The study of intracellular protein interactions has been challenged by the ability to efficiently capture and preserve protein complexes, especially when attempting to isolate weak or transient interactions. In a recent webinar Rob Chumanov took us through techniques used to study proteins in their native environment and highlighted the most efficient method for studying them based on the HaloTag® covalent tag.
The older generation of protein tags is not ideal for studying protein interactions. These routine protein tags have been adapted for specific narrow applications, such as GFP for live-cell imaging and epitope tags (His, FLAG, and GST) for both fixed-cell imaging and capture of protein:protein interactions. As a consequence, often researchers create multiple protein fusion constructs with different tags in order to optimally characterize protein function. In contrast, HaloTag® technology provides broad flexibility for both imaging and biochemical applications with a single tag that binds rapidly, covalently, and specifically to synthetic small molecule ligands that ultimately determine the functionality of HaloTag®. Continue reading “How to Identify Physiologically Relevant Protein Interactions Using Covalent-Capture HaloTag(R) Technology Information”
Efficient proteolysis is a major requirement for protein mass spectrometry analysis. Incomplete digestion has multiple ramifications including decreased number of identified proteins, compromised analytical reproducibility and protein quantitation, etc. Trypsin is one of the most robust proteases and is characterized by efficient proteolysis. Typical trypsin reactions do not digest proteins to completion, missing 15–30% of cleavage sites. Incomplete digestion affects protein identification, reproducibility of mass spectrometry analysis and accuracy of protein quantitation. Supplementing Trypsin with Lys-C compensates for the majority of missed cleavages. Continue reading “Enhanced Protein Mass Spectrometry Analysis with Trypsin/Lys-C Mix”
Quantitative PCR (qPCR) and reverse transcription qPCR (RT-qPCR) are useful tools in laboratories around the world for absolute or relative quantification of DNA or RNA targets from biological samples. Once you understand the basic principles and have optimized the reaction and cycling parameters, these techniques tend to be rapid, accurate and easy-to-perform—makes me wish that qPCR and RT-qPCR were more commonplace when I worked in the lab and used radioactive Northern blots to quantitate my gene of interest. This is clearly another case of “If only I had known then what I know now”. While I’m no longer working in the lab, it’s not too late for you to benefit from a good introduction to qPCR and RT-qPCR, and on January 15, 2013, that’s exactly what one Promega Research and Development Scientist presented: A webinar entitled “Optimize Your qPCR and RT-qPCR Assays with Careful Planning and Design”. Continue reading “Harnessing qPCR and RT-qPCR in Your Laboratory”