Scientists have had a love-hate relationship with PCR amplification for decades. Real-time or quantitative PCR (qPCR) can be an amazingly powerful tool, but just like traditional PCR, it can be quite frustrating. There are several parameters that can influence the success of your PCR assay. We’ve highlighted ten things to consider when trying to improve your qPCR results.
Continue reading “Top 10 Tips to Improve Your qPCR or RT-qPCR Assays”Today’s blog is from guest blogger Ken Doyle of Loquent, LLC. Here, Ken reviews a 2014 paper highlighting specific considerations for using reporter assays to study miRNA-mediated gene regulation.
The accelerated pace of research into noncoding RNAs has revealed multiple regulatory roles for microRNAs (miRNAs). These diminutive noncoding RNA species—typically 20-24 nucleotides in length—are now known to mediate a broad range of biological functions in plants and animals. In humans, miRNAs have been implicated in various aspects of development, differentiation, and metabolism. They are known to regulate an assortment of genes involved in processes from neuronal development to stem cell division. Dysregulation of miRNA expression is associated with many disease states, including neurodegenerative disorders, cardiovascular disease, and cancer.
Typically, miRNAs act as post-transcriptional repressors of gene expression, either by targeted degradation of messenger RNA (mRNA) or by interfering with mRNA translation. Most miRNAs exert these effects by binding to specific sequences called microRNA response elements (MREs). These sequences are found most often within the 3´-untranslated regions (3´-UTRs) of animal genes, while they may occur within coding sequences in plant genes.
Studies of the regulatory roles played by miRNAs often involve cell-based assays that use a reporter gene system, such as luciferase or green fluorescent protein. In a standard assay, the reporter gene is cloned upstream of the 3´-UTR sequence being studied; this construct is then cotransfected with the miRNA into cells in culture. A study by Campos-Melo et al., published in September 2014, examined this experimental approach for miRNAs from spinal cord tissues, using firefly luciferase as the reporter gene and Renilla luciferase as a transfection control. Continue reading “A Normalization Method for Luciferase Reporter Assays of miRNA-Mediated Regulation”
One piece of advice you will get from our Technical Services and R&D Scientists with regard to cell-based assays is to pay attention to what you are doing. Sounds obvious, but sloppiness can easily enter into the equation. Do you always count your viable cells with a hemocytometer and trypan blue exclusion before you split a culture? Do you always make sure that each well of your plate or plates contain the same number of cells? Two of our scientists, Terry Riss and Rich Moravec, published a paper demonstrating how decisions you make in experimental setup can ultimately affect the results you obtain. A natural consequence of this is difficulty replicating experiments if you didn’t pay attention to the details during the initial experimental setup.
Cell Density Per Well Affects Response to Treatment
To demonstrate how cell density can affect your data, Riss and Moravec set up parallel plates with three different cell densities of HepG2 cells and measured the response to tamoxifen. The lower the cell density per well, the more pronounced the effect of the tamoxifin on the cells. Higher density cells were more resistant to tamoxifen. Continue reading “Take Notes and Graduate Faster!”
Q: What is the easiest way to clone PCR Products?A: The simplest way to clone PCR Products is to amplify the product using thermostable polymerases such as Taq, Tfl or Tth polymerase. These polymerases add a single deoxyadenosine to the 3´-end of the amplified products (3´-end overhang), and can be cloned directly into a linearized T-vector.
A: To clone PCR products that have been amplified with a polymerase that have proof reading activity into a T-vector, you will need to perform an A-tailing step using Taq DNA polymerase and dATP. Blunt ended restriction digest fragments can also be A-tailed using this method. The method below uses GoTaq Flexi DNA Polymerase (comes with a Mg-free reaction buffer), but any Taq DNA polymerase can be used.
Set up the following reaction in a thin-walled PCR tube:
1–4µl purified blunt-ended DNA fragment (from PCR or restriction enzyme digestion)
2µl of 5X GoTaq Reaction Buffer (Colorless or Green)
2µl of 1mM dATP (0.2mM final concentration)
1µl GoTaq Flexi DNA Polymerase (5u/µl)
0.6µl of 25mM MgCl2 (1.5mM final concentration)
Nuclease-free water to a final volume of 10µl
Incubate at 70°C for 15–30 minutes in a water bath or thermal cycler.
A: T vectors are linearized plasmids that have been treated to add T overhangs to match the A overhangs of the PCR product. PCR fragments that contain an A overhang can be directly ligated to these T-tailed plasmid vectors with no need for further enzymatic treatment other than the action of T4 DNA ligase.
For a complete PCR Cloning protocol, Visit the Cloning Chapter of the Promega Protocols and Applications Guide.
Many studies, from reporter assays to protein localization to BRET and FRET, require successful transfection first. Yet, transfection can be tricky and difficult. There are many considerations when planning transfection of your cells including reagent selection, stable or transient experiment, type of molecule and endpoint assay used. Here we discuss these considerations to help you plan a successful transfection scheme for your experimental system. Continue reading “General Considerations for Transfection”
Updated 02/12/2021
Transient transfection is often used to perform reporter assays. We have advocated using a dual-reporter system for decades to normalize the data obtained and gain a clearer understanding of your results. The experimental reporter should vary with treatment and the control reporter should vary little with treatment. The control reporter thus serves as a marker to help you understand the relative activity of your experimental reporter. The bioluminescent Dual-Luciferase® method allows for sequential detection of the second reporter in a single sample providing a simple two-step normalization method. Here are seven ways in which dual-reporter assays help you avoid misinterpreting results.
Simply comparing the ratio of the experimental to the control reporter can resolve differences in:
Bonus Benefit from Dual-Luciferase®, Dual-Glo® and the NanoGlo® Dual Luciferase Reporter Systems: No Lysate Splitting: Promega dual-reporter assays are designed for same-well multiplexing so there is no chance of variations creeping into your data due to unequal splitting of the cellular lysate to measure two separate reporter activities.
Since the introduction of the first bioluminescent dual-luciferase assay in 1995, this approach has been used in countless studies to advance our scientific understanding of cellular gene regulation. To learn more about the last 30 years of bioluminescent innovations and research discoveries please visit our 30th anniversary web page.
Further Reading:
Normalizing Genetic Reporter Assays Approaches and Considerations for Increasing Consistency and Statistical Significance
Before you begin your subcloning, you need to know: The restriction enzyme (RE) sites available for subcloning in your parent vector multiple cloning region (or in the insert if you need to digest the insert); the RE sites available in the destination vector multiple cloning region (MCR); and if these same sites also occur in your insert. Once you know this information, you can use the chart below to decide which subcloning strategy to use.
![4498MA-[Converted]](https://www.promegaconnections.com/wp-content/uploads/2014/10/4498MA-Converted-e1413386878386.jpg)
To learn more about subcloning, visit our Subcloning Notebook.
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 “To inject or not inject?”
Proteomics, the analysis of the entire protein content of a living system, has become a vital part of life science research, and mass spectrometry (MS) is the method for analyzing proteins. MS analysis of protein content allows researchers to identify proteins, sequence them and determine the nature of post translational modifications.
Mass spectrometry allows characterization of molecules by converting them to ions so that they can be manipulated in electrical and magnetic fields. Basically a small sample (analyte) is ionized, usually to cations by loss of an electron. After ionization, the charged particles (ions) are separated by mass and charge; the separated particles are measured and data displayed as a mass spectrum. The mass spectrum is typically presented as a bar graph where each peak represents a single charged particle having a specific mass-to-charge (m/z) ratio. The height of the peak represents the relative abundance of the particle. The number and relative abundance of the ions reveal how different parts of the molecule relate to each other.
For the study of large, organic macromolecules, matrix associated laser desorption/ionization (MALDI) or tandem mass spec/collision induced dissociation (MS/MS) techniques are often used to generate the charged particles from the analyte. MS analysis brings sensitivity and specificity to proteome analysis. The technique has excellent resolution and is able to distinguish one ion from another, even when their m/z ratios are similar. Macromolecules are present in extremely different concentrations in the cells, and MS analysis can detect biomolecules across five logs of concentration.
Continue reading “Monitoring Mass Spec Instrument Performance and Sample Preparation”