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Past Issue in 2013
Volume: 3, Issue: 7
Cell Biology
Micrococcal Nuclease (MNase) Assay of Arabidopsis thaliana Nuclei
Micrococcal nuclease (MNase) is able to produce double-strand breaks within nucleosome linker regions. The efficiency of MNase digestion depends on the degree of chromatin compaction, being more easily digested the regions of less compacted chromatin. The MNase protocol described here can be used to asses changes in the chromatin structure of nuclei extracted from Arabidopsis seedlings.
Immunology
Allergen Sensitization and Challenge to Ovalbumin
This protocol describes the sensitization and challenge of mice with ovalbumin for use as an acute murine model of asthma. This protocol induces reproducible airway inflammation and remodelling, and bronchial hyperresponsiveness to methacholine as measured by barometric plethysmography, as well as by the Flexivent® technique in Balb/c mice.
Measurement of Airway Responsiveness in the Anesthetized Mouse
Airway hyperresponsiveness to methacholine is an important characteristic of asthma. Many devices can be used to measure airway responsiveness in the mouse but it is well established that the invasiveness of a measurement technique is correlated with its precision and reproducibility. This protocol describes how to measure airway responses to aerosolized methacholine in anesthetized, tracheotomized, and mechanically ventilated mice using the forced oscillation technique with FlexiVent® (SCIREQ). This is a computer-controlled precision piston pump that can intersperse mechanical ventilation with volume and pressure controlled manoeuvres to obtain accurate, reproducible measurement of respiratory mechanics.
Microbiology
Neutral Red Assay for Murine Norovirus Replication and Detection in a Mouse
Neutral red (NR) is a dye that must be actively imported into the cell, and, therefore, the dye has been used for decades to selectively stain living cells. In addition, NR can also be incorporated into virus particles, although the mechanism behind this is poorly understood. Once encapsulated into the virion, NR, a light sensitive dye, can be photoactivated to inactivate the virus. The proposed mechanism explaining this observation is that activation of NR allows the dye to cross-link viral genome to viral capsid and thus preventing viral uncoating and infection. To study the early events of murine norovirus (MNV)-host interaction, light-sensitive NR-containing MNV is used to distinguish between input virus (i.e., NR-containing virus) and replicated virus (i.e., NR-free virus). This protocol describes the incorporation of NR into MNV capsids and the use of these virions for detection of viral replication in a mouse and in tissue culture by standard plaque assay. The same technique is also used for study of poliovirus replication (1-3). Thus, there is the potential that this technique can be used for additional non-enveloped viruses. However, this has to be tested on a case-by-case basis as unpublished data on feline calicivirus suggests not all viruses may be able to stably incorporate NR into their capsid (J. Parker, personal communication).
Fusarium Virulence Assay on Wheat and Barley Seedlings
Fusarium root and crown rot is a very important complex disease of small grain cereals worldwide which may lead to very high yield losses. Traditional virulence assays are time consuming and often require plants to be grown in greenhouses or climatic chambers in soil. We describe a rapid laboratory assay for assessing such a disease in wheat and barley seedlings. The method could be successfully used for testing fungal virulence as well as to assess plant resistance.
HIV-1 Single Cycle Infection
The role of a viral or cellular protein on HIV-1 infection can sometimes be difficult to assess in a system where the virus is able to replicate for several cycles. Indeed, some effects are only observable at early time points, and can be masked (or on the contrary, artificially increased) after several rounds of viral replication. Therefore, to clearly know at which step of HIV-1 replication cycle one protein acts, it is important to be able to study one cycle of infection only. This protocol allows rapid and robust quantification of HIV-1 single cycle infection, and can be used to compare mutant viruses, or treatments with different drugs.
Quantification of HIV-1 DNA
The reverse transcription (RT) reaction is a critical step in HIV-1 life cycle. It is very strongly regulated and the target of several restriction factors (TRIM5α, APOBECs, SAMHD1, etc.). The progress of reverse transcription can be followed by measuring viral DNA by quantitative PCR (qPCR). This method is sensitive enough to allow detection of low amounts of HIV-1 DNA in infected cells and discriminate between several types of reverse transcription intermediates (so called 《early》 and 《late》 RT products, 2 Long Terminal Repeat (LTR) circles, integrated DNA).
Molecular Biology
Northern Blot of tRNA in Yeast
tRNAs are small RNAs around 70-90 nt. tRNAs are different from many other small RNAs in that they are very abundant, which makes it difficult to study their transcriptional regulation by traditional northern blot. Traditional Northern blot involves incorporation of radioactive nucleotides through polymerization, however, tRNA is too short for polymerization. Traditional Northern blot detects changes in RNA levels, however, tRNA are so abundant that small changes in their levels will escape detection. For these reasons, metabolic labeling by radioactive Uracil has been used instead. However, metabolic labeling can only examine changes in total tRNA, but cannot distinguish different types of tRNAs. The following protocol describes a method to examine individual tRNA gene transcription by northern blot.
Western Analysis of Histone Modifications (Aspergillus nidulans)
Western blotting allows for the specific detection of proteins and/or modifications of proteins by an antibody of interest. This protocol utilizes a crude nuclei extraction protocol for Aspergillus nidulans to enrich for histones and other nuclear proteins prior to gel electrophoresis. Post translational modifications of histones may then be easily detected. After electrophoresis, the selected antibodies are used to detect and quantify levels of the modifications of interest.
Metabolic Labeling of Yeast RNA with Radioactive Uracil
To examine gene expression, Northern blot or Real-Time PCR can be used to detect low abundant RNA such as mRNA. However, for high abundant RNAs such as rRNA and tRNA, Northern blot will not be able to discriminate the newly synthesized RNA from total RNA. Therefore, metabolic labeling is necessary to evaluate the expression of rRNA and tRNA genes. In this protocol, I describe a step-by-step method for labeling yeast RNA with radioactive uracil and examine the synthesis of these high abundant RNAs.
Neuroscience
Fear Conditioning Assay in Mouse
The study of fear memory is important for understanding various anxiety disorders in which patients experience persistent recollections of traumatic events. These memories often involve associations of contextual cues with aversive events; consequently, Pavlovian classical conditioning is commonly used to study contextual fear learning. A form of contextual fear conditioning that is becoming increasingly important as an animal model of anxiety disorders uses predator odor as a fearful stimulus. Innate fear responses to predator odors are well characterized and reliable; however, attempts to use these odors as unconditioned stimuli in fear conditioning paradigms have been highly dependent on experimental setup and have produced inconsistent behavioral results. Here we present a contextual fear conditioning paradigm using coyote urine as the unconditioned stimulus, which has been shown to produce consistent contextual freezing in response to fear learning (Wang et al., 2012).
Plant Science
Northwestern Blot of Protein-RNA Interaction from Young Rice Panicles
The northwestern assay is employed to study the interaction between protein and RNA. The RNA binding proteins tend to bind to different kinds of RNA through either known domains or unknown sequences of proteins. Rice LGD1 recombinant protein, a grass-specific novel protein with RNA binding sequences in its C-terminal, was used to probe its function as an RNA binding protein. The LGD1 comprising von Willebrand factor type-A domain (vWA), coiled-coil and nuclear localization signal (NLS) is a class of protein that localizes both in the nucleus and cytoplasm. Although LGD1 does not contain any putative RNA binding domains, we could find high-affinity RNA binding residues at the C-terminus using ‘RNABindR’ prediction software (Terribilini et al., 2007). The LGD1 recombinant protein, purified from bacteria, somehow forms both dimer and monomer even under denaturing conditions. However, only the dimeric form is able to bind to total and mRNAs. Due to its reproducibility and reliability, we believe that this protocol can be used across different organisms.