...melting profiles (Dwight, 2011).  Reference Genes  Choosing a set of reference genes deserves more attention...attention than it usually receives. Different reference genes have shown to have as much as sixfold variation...of Connect Four! Image created in BioRender.   Passive Reference Dyes Whatever your pipetting method...occur. A passive reference dye, such as ROX, can help normalize for that. Passive reference dyes are inert...Liu and Saint No Raw Data Reference and target genes can have different efficacies DART-PCR No ...will soon become a qPCR master. References and Resources  References Biassoni R; Raso A. (eds.), Quantitative...mRNA, which can be used as a proxy measure for differences in protein expression. (If you're just getting...

...heterogeneous population, cells behaving differently (e.g. different growth properties, migration abilities...targeting all differentially expressed genes that are known drug targets, or all differentially expressed ... by choosing which “Reference Library” you’d like to use in your screen. Currently, eleven published libraries...best gRNAs by the depositing lab. The different labs use different algorithms to calculate which would ...favorite Reference Library, for example related to strategies beyond SpCas9, or species not currently included...were not found in the reference library. It’s not uncommon that genes have different names, and the software...generation sequencing to identify which gRNAs are differentially expressed in the separate groups and, as a ...

...For CD365, they examined two different antibody clones produced by different vendors. The specific epitope... panels, where different manufacturers may have the same clone available with different conjugates. Summary...commercial antibody manufacturers. References and resources References Ayoubi, R., Ryan, J., Biddle, M. ...include a discussion of the Human Leucocyte Differentiation Antigens (HCDM) workshop approach, which focuses...one size fits all. The approaches used will be different depending on the sample and target type. To start...an approach that prioritizes more reproducible/renewable reagents, such as recombinant or hybridoma-derived...(e.g. DepMap) – you’ll need to ensure that the parental cell lines of your chosen KO line are likely to...

...most organisms on Earth, but different organisms have different preferences for the codons they use to ...have different codon usage biases? The reasons for varied codon preferences among organisms aren’t completely...poorly charged tRNAs, different regions of the protein may be translated at different rates. The ribosome...Organisms often need to express genes at different levels under different conditions. With varied codon usage...constraints have molded which codons are used preferentially in which organisms - organisms have codon usage...your organism of choice. Unfortunately, codon preferences make it so you cannot choose among the possible... evolutionary constraints that lead to these preferences and what can we do about them? Read on to find...

... directed differentiation To understand how each human TF can contribute to differentiation, the Zhang... scientist at [email protected]! References and Resources  References Joung J. et al., A transcription...patterns determine everything from stem cell differentiation fate to tumor suppression (and most everything...construct. Cells were cultured following a differentiation protocol before performing single-cell RNA ...embryonic stem cells. Transduced stem cells were differentiated and single cell RNA-seq was performed to map...    This atlas linked TF expression to differentiation outcomes as well as global transcriptional ...program changes from this screen successfully differentiated cells resembling all three germ layers (ectoderm...

...reflect massive differences in protein abundance. Validating the assumptions for different loading controls...detection range be linear! Resources and References References Aldridge, G. M., Podrebarac, D. M., Greenough... that lanes are loaded evenly. Created with BioRender.com. Loading controls are even more important...entire blot. So how can you accurately compare different lanes? In relative quantification, the POI bands... the two conditions are equal. Created with BioRender.com. This kind of normalization is a nearly ...with separate controls (green). Created with BioRender.com. Researchers often rely on assumptions that...normalize your results, you are assuming that all differences in the loading control are due to loading and...

...organism. Output diversity: Different tools provide results to the user in different formats. This can have...the piñata beating party goers are altogether different. They are filled with blind anticipation. Their...spill to the ground. There is something akin to a frenzy as the crowd tries to weigh and determine the value...response; and, it is the stage I think we are currently at in the CRISPR software space. The CRISPR research...year.   Breaking down the barriers: My view on current sgRNA design tools The goal of this post is to ... sgRNA and represents the lion’s share of the currently available tools (we will focus on these tools ...required to fully evaluate off-targeting, but currently no software has been designed for this purpose...

... with biorender.com.   Of course, this is quite easy to do on a plot generated in Biorender! While...with that…and happy flowing!    References and resources  References Alyamani, A., Kalamegam, G., Ahmed...read from Marker A and Marker B. Created with biorender.com.   The goal of flow cytometry is to use ...usually tell where this point is by looking at the different populations. In the case of Figure 2, one population...and one population that is B-. Created with biorender.com.   If it’s hard to conceptualize where the...   Figure 3: It's often easier to identify different populations by imagining (or drawing) a quadrant...population and everything outside the gate is a different population.  Gating and plot orders  As the gates...

...utilized mice to study how different mutations in leukemia impact different treatment regimens (Zuber ...β-cells that originate in different regions of the pancreas and produce different levels of insulin (Hesselson...1970s when Sydney Brenner brought it into the lab to study neuronal development (Brenner, 1973). C. elegans... developed for emerging model organisms! References Brenner, Sydney. "The genetics of Caenorhabditis ...injecting mice with retroviral vectors that harbored different mutations found in leukemia, the Lowe lab was ...development studies as their embryos are completely transparent. Thus scientists can easily observe early developmental...can be difficult in other vertebrate species. Transparency also allows scientists to easily observe fluorescently...

...research program (think a subtle difference in mouse chow, a different water filtration system, environmental...were doing something differently. Communication comes in many forms—from transparency about data to allow...make for a transparent and collaborative research environment. Journals as enforcers Currently, most of ...original paper that everyone in your lab has been referencing for the last 10 years, and everything will be...original researchers actually did. Admittedly, the differences are often very subtle, and it can take a lot ...of the burden for pushing for more transparency in science communication falls on the shoulders of journals...developed STAR Methods to improve the structure and transparency of methods sections, including clear information...

... well as predict the channelrhodopsin’s photocurrent strength, wavelength sensitivity, and off-kinetics...one region of the brain, which is needed since current channelrhodopsins tools have low conductance, but...activation to a small volume of brain tissue (~1 mm3). Current channelrhodopsins also need high-intensity light... neuron and causes it to fire. Created with BioRender.com. To make channelrhodopsin-based optogenetics...sensitive and have enhanced conductance compared to current channelrhodopsins. Their new channelrhodopsins,...engineering channelrhodopsins to have improved photocurrent properties: The sequence and structures that...electrophysiology, a technique that’s used to measure electric current of ion channels in living cells. Multiple properties...

... immune system make different isotypes of antibodies that recognize many different epitopes of the target... and protein G each have different affinities for IgG subtypes from different animals so it’s important...variability in the strength of staining and background noise. If you do use different lots of your polyclonal...successful next experiment!     References and Resources References Ascoli CA, Aggeler B (2018) Overlooked... cover polyclonal antibodies, one of the many different types of antibodies available (others you’ll encounter...its serum several weeks later. Created with biorender.com. To generate polyclonal antibodies, an animal...polyclonal antibodies from the serum There are a few different ways the antibodies can be isolated from the serum...

...detergent like RIPA. You can try a few different lysing buffers and strengths. If that’s not working, consider...Membrane pore size Membranes come in different pore sizes for different proteins: try using a 0.2 µm (psi...Protein not properly lysed Different lysing buffer/adjust lysing buffer strength Sonication based lysing...Best of luck and happy gradients!  References and resources References Luo, H., Rankin, G., Straley, S.,... the antibody’s dynamic range. Created with biorender.com   Alternatively, you can run a reagent gradient...cut up the membrane to treat each lane with a different concentration of your reagent. Be aware that this...high signal with low background. Created with biorender.com   Both gradients can be informative, so ...

...find lots of different plasmids containing the same protein of interest tagged with different fusion proteins...you the flexibility to choose different colors or ligands for different experiments without having to ...intracellular recordings, switching between different colored ligands at different time points.   Figure 3:...or HaloTag have been shown to perform differently for different ligands (Wilhelm et al., 2021). While ...things up for your next experiments! References and resources References Bottanelli, F., Kromann, E. B., Allgeyer...proteins for different experiments over the years — a green fluorescent protein, a red fluorescent protein...binds a synthetic chloralkane (CA) ligand. The current standard version known as HaloTag is actually HaloTag7...

...Created with BioRender.com. For specific prime editing tools There are a lot of different prime editing...offer two different strategies for protecting pegRNAs from degradation. Created with BioRender.com. ...software in the comments below! References and Resources References Anzalone, A. V., Randolph, P. B.,...Figure 1: Prime Editing schematic. Created with BioRender.com. The pegRNA specifies both the genomic location... These tips are a great starting point: Test different lengths of the primer binding site, starting with...still be optimized (Anzalone et al., 2019). Test different lengths of reverse transcriptase template, starting... templates, it is even more important to test different lengths and sequences, as unintended secondary...

...horizon (Altae-Tran et al., 2023)! References and Resources References Abudayyeh, O. O., Gootenberg, J. ...probably still edit genomes. Understanding the different types of CRISPR systems can be as confusing as...application of CRISPR-Cas9 kicked off over a decade of frenzied interest in bacterial and archaeal immune systems...multi-pronged analysis to sort effectively. The current classification system relies on a combination of...effector may have the same name but be completely different! They are merely linked by a similar function....typically based on species of origin. There are currently two classes, six types, 33 subtypes, and 17 variants...to variant (shown in italics). Created with BioRender.com.   Class 1 versus class 2 There are two ...

... attend conferences. Depending upon your discipline, there are different types of conferences that you...organizations that hold large, annual conferences. Gordon conferences offer more focused and discipline-specific...experiences, and several Gordon conferences are held every year in different locations all over the world...employer’s career pages to look for current job postings. You may then reference the connection you made at the...were so critical to me landing my own position! Conferences One of the single most important things you can...world. But simply attending the conference is usually not enough. How do you stand out from the hundreds ...people. Attend smaller talks within the bigger conference, especially ones that are more career-focused...

... events (see Jin & Maren, 2015). Directional flow of information between different parts of the brain ...Castle et al., 2014). Differences between AAV serotypes may be also linked to differences in viral uptake by...these different AAV vectors into a brain region, the AAV particles will infect neurons at different ratios...architecture, in particular the connections between different brain regions. For example, communication between...forming a neuronal loop which is activated and strengthened during memory formation and memory retrieval... for degradation. These two pathways also use different cytoskeletal machineries to facilitate transport...while anterograde transport relies on kinesin. Different rates of speed for various forms of retrograde...

...test assesses the sum of differences between pairs). Samples arising from different subjects or populations...significant differences between the means of your groups. However, it will only tell you if differences exist...samples are different from each other, or if every permutation of pairs within the five is different. To identify...data – and it looks great! There are some exciting trends in your experimental groups, but how do you determine...groups to determine if they are statistically different. They are classic parametric tests and one of ... whether or not two samples are statistically different than one another, a two-tailed test should be ...control group. If you only care if one group is different in a certain direction (i.e., you just want to...

...immunodetection tools is their strong signal strength. This signal strength is due in large part to their larger...primary antibodies raised in different species, or at least from different IgG subclasses, so that secondary...incubation of 1° and 2° Must use different species and/or different IgG subclasses of 1° for multi-color...limited cross-reactivity between different IgG subclasses and different species, nanobodies are useful ...mouse or rabbit variety. While antibodies certainly aren’t “broken,” their production does require continued...Multiple polyclonal secondary IgGs can bind to different epitopes of a primary antibody. B) Each arm of...Monoclonal Only anti- rabbit and -mouse versions are currently available Not commercially available Small...

...output is measured and quantified. Created with BioRender.com.   Optogenetic tools Types of control We...excitatory and inhibitory channels activated at different wavelengths of light (Vierock et al., 2021). Step-function...stay on until a second pulse of light, with a different wavelength, turns it off, allowing the cell to... secondary signaling pathways. There are many different opsin (natural and engineered proteins responsive...against the concentration gradient. Created with BioRender.com.   Opto-G-protein coupled receptors  G-protein...intracellular domain of an endogenous GPCR, such as adrenergic receptors. When exposed to light, there is a ...pocket and activates the receptor. Created with BioRender.com.   Controlling secondary pathways with photoswitches...

...unaccounted for, may lead to different results. Different setting, different population, new genetic variation...in the process of sampling and inference-making. Even perfect transparent and rigorous studies will give...obtaining a different result after conducting a replication study. An important difference between the...are investigated to look for unexpected trends or differences between groups. Hypothesis generation is...images, measuring the main outcomes in several different ways, and other questionable research practices...variation, or different handling can all be important.  If we eliminate the problems above: unclear methods...previously unknown variable actually makes a big difference for the robustness of the findings.   How do...

...arbitrate for them. They were different people with some very different opinions, and some of them would...that there are different types of conflict. Most people perceive any conflict as inherently bad, and in ...actually been a meeting? Different teams frequently prefer to communicate in different ways, so the source ...thomas-kilmann-conflict-resolution-model/    References and Resources References Thomas-Kilmann Conflict Model, Management...without my “neutral” presence to alleviate those differences, they had a hard time maintaining their friendships...natural inclination for this skill that you could strengthen. The right set of transferable skills, coupled...hypothesis stronger. When you share data at a conference poster session, you don’t necessarily want everyone...

...successful IF experiment.    References and resources References Im K, Mareninov S, Diaz MFP, Yong WH (2018...target primaries produced in different species and to label targets with different colors. Multiplex immunofluorescence...Speaking of different colors, having a palette of secondary antibodies in a few different species and ...dimensional epitopes. Different sample processing steps can alter epitopes in different ways, so antibodies...indirect immunofluorescence There are a couple different approaches to be familiar with when talking about...immunofluorescence work. Image created with BioRender.com   Indirect IF is a much more common approach... By using multiple primary antibodies made in different species on a single sample, you can then use the...

...PMCID: PMC3929550. 3. Kaati, Gunnar, Lars O. Bygren, and Soren Edvinsson. "Cardiovascular and diabetes mortality...mortality determined by nutrition during parents' and grandparents' slow growth period." European Journal...in the body contain the same DNA but have very different structure and function, and can have a specialized...were experienced in a period of life when the grandparents’ gametes were developing, an epigenetic change...to have occurred that was passed down to the grandchildren; in this study,epigenetic changes were statistically...syndrome and Prader–Willi syndrome, which have very different disease phenotypes, are two fascinating examples...the gene is expressed in normal individuals (in a parent-of-origin-specific manner), but the expression ...

...converted to a different isotype, then two different secondary antibodies can be used with different fluorescent...have different binding properties (Morelock 1994). While in the majority of cases the differences are ...their parental species which can be bound by broadly reactive secondary antibodies to that parental species...conversions and chimeric antibodies! References and resources References Liddell, E. (2013). Antibodies. In...isotype conversion process the variable regions of a parental antibody (blue, rat) are cloned into a backbone...changing an antibody’s natural isotype to that of a different group (Fig. 1). This process, called isotope conversion...antibody’s isotypes, however, you could then use 2 different secondaries for your antibody pair and visualize...

...demonstrated that different deaminase and UNG enzymes had different sequence preferences, which they defined...outcomes of their various CGBEs at different target sequences and in different cell types, available at www....component of the nucleoside inosine. Created with BioRender.com. Although it was a huge innovation, there... Figure 2: CRISPR R-loop. Created with BioRender.com. Base editors take advantage of this single-stranded...non-target strand.” To avoid confusion, we usually use different terminology when discussing base editors, like...up to 68% at the most favorable target sites. Differential cytosine editing between bacteria and mammalian...sites. But the same strategy in E. coli produced different results. GBEs seemed to follow the same initial...

...activated cell sorting (FACS) to isolate differentiated from undifferentiated cells at the end of the screening... to study neuronal differentiation, it could also be applied to the differentiation of other CNS cell ... promoters (aCaMKII and hSyn). Created with BioRender.com. Before using gRNAs in vivo, they should...expression of dCas9-VPR (bottom). Created with BioRender.com. The Day Lab used their optimized vectors...to mRNAs generated from the same gene but with different untranslated or coding regions. Typically, each...straightforward to use CRISPRa to selectively target these different transcriptional start sites in order to better... expression of Bdnf resulted in hundreds of differentially expressed genes between Bdnf I-overexpressing...

...emphasizing the antibody’s specificity. Strength and accuracy The strength is based on binding specificity and...fear of referencing a wrong section. The antibody screening and development of a focused reference bring...School of Medicine at Mount Sinai.  References and resources References Baker M. Reproducibility crisis: ...), variability (separate batches can perform differently – happens when the antibody is produced from ...a new set of animals), and wrong application (different experiments/conditions can change binding ability...off-target molecules), reliable (wouldn’t perform differently over time), compatible with the desired applications... developed a criterion that includes accuracy, strength, and reproducibility. We test the antibodies on...

...can recruit parents from any background, it really helps if activities are run by parents who can answer...teachers reached out to their students’ STEM-oriented parents looking for experts to come give guest presentations... and technology into the school curriculum. Our Parent Teacher Association (PTA) has been instrumental... the annual Math & Science Night, a chance for parents and other members of the community to share their...you live in a school district that has a lot of parents in STEM careers, chances are they’re wondering ...expertise with our district middle school was a very different experience since, unlike elementary schools, middle...teachers at our school put out the call to local parents who would feel comfortable speaking on topics that...