Bisulphite conversion protocol
A standard protocol for the conversion of 2 &mu;g of genomic DNA is described below. Smaller or larger amounts of genomic DNA (100 pg-200 &mu;g) can also be bisulphite treated successfully. These reaction conditions result in complete conversion (99.5-99.7%) of almost every target DNA sequence3.
1. DNA Preparation
Prepare samples by incubating genomic DNA with bisulphite DNA Lysis Buffer (2 &mu;g tRNA, 280 ng/&mu;l Proteinase K, 1% SDS) in a total volume of 18 &mu;l for 1 hr at 37&deg;C. Note: This is important to achieve maximal bisulphite conversion, especially with DNA isolated from clinical samples where there may still be protein associated with the DNA.
2. DNA Denaturation
<ol>
	<li>Denature 2 &mu;g DNA in a volume of 20 &mu;l by adding 2 &mu;l of freshly prepared 3M NaOH to a final concentration of 0.3M.</li>
	<li>Incubate the samples at 37&deg;C for 15 min in a water bath, followed by incubation at 90&deg;C for 2 min in a heat block. Immediately place the tubes on ice for 5 min.</li>
	<li>Centrifuge the tubes at 4&deg;C for 10 s at 10,000 g, to ensure the DNA is at the bottom of the tube.</li>
</ol>
3. Bisulphite Deamination
Sulphonation &amp; Hydrolytic Deamination
<ol>
	<li>Prepare fresh solutions of 10 mM Quinol and saturated sodium metabisulphite pH 5.0 (7.6 g Na2S2O5 with 464 &mu;l of 10 M NaOH, made up to 15 ml with water). The solution of saturated sodium bisulphite is achieved by gently inverting the reagent/H2O mixture, with minimum mixing and aeration. If necessary, adjust the pH with 10 M NaOH, for full dissolution of the metabisulphite. Note: As it is a saturated solution, small lumps may still remain undissolved.</li>
	<li>Add 208 &mu;l of the saturated metabisulphite and 12 &mu;l of 10mM Quinol to the denatured DNA (20 &mu;l), in a final volume of 240 &mu;l to a final concentration of 2.31M bisulphite/0.5mM Quinol, pH 5.0. Gently mix all reagents and centrifuge for 10 sec to ensure all of the droplets are at the bottom of the tube.</li>
	<li>Overlay the samples with 200 &mu;l of mineral oil. Incubate the samples at 55&deg;C in a water bath, for 4-16 hrs. The length of bisulphite treatment is dependant on the quantity and quality of the DNA to be converted. For DNA of poor quality or degraded DNA, limit the incubation time to 4 hrs. Note: it is important that that the bisulphite conversion takes place in the dark to avoid oxidation, so if that is not possible wrap the tubes in foil prior to incubation.</li>
	<li>At the end of the appropriate incubation time, spin the tubes briefly in a microcentrifuge to ensure all the liquid is at the bottom of the tube.</li>
	<li>Recover the bisulphite treated DNA from under the oil layer by carefully pipetting out the DNA solution from the bottom of the tube, without taking up any of the mineral oil.</li>
</ol>
Desalting
<ol start="5">
	<li>Remove any free bisulphite ions by passing through a desalting column and eluting in 50 &mu;l of milli-Q water (MQH2O). Depending on the quantity and quality of the genomic DNA and how it is prepared, different desalting columns can be used. We routinely use Promega Wizard Clean up columns, as these columns are suitable to remove the SDS used in the preparation of the DNA prior to conversion.</li>
</ol>
Alkali Desulphonation
<ol start="6">
	<li>The bisulphite adduct is removed from the uracil ring by desulphonation. Desulphonate by adding 5.5 &mu;l of freshly prepared 3M NaOH to a final concentration of 0.3M, then incubate the samples at 37&deg;C for 15 min.</li>
	<li>Centrifuge briefly and add 1 &mu;l of tRNA (10 mg/ml).</li>
	<li>Neutralize the solution by the addition of 33 &mu;l of ammonium acetate (NH4O Ac), pH 7.0 to a final concentration of 3M.</li>
	<li>Ethanol-precipitate the DNA by adding 330 &mu;l of ice cold 100% ethanol, mix well by inversion. Leave at -20&mu;C for 1 hr to overnight. Centrifuge at 14,000 g for 15 min at 4&deg;C. Remove all traces of supernatant and air dry the precipitated DNA for ~20 min.</li>
	<li>Re-suspend the DNA pellet in 50 &mu;l/&mu;g of 0.1 TE (10mM Tris-HCL, 0.1mM EDTA, pH 8.0) or H2O. Leave at room temperature for approximately 2hrs. Occasionally vortex the tubes to ensure the DNA is dissolved, followed by a quick centrifuge.</li>
	<li>Use immediately for PCR amplification, or store at -20&deg;C for 1-10 years depending on the quantity and quality of DNA.</li>
</ol>
4. PCR Amplification
Primer Design
<ol>
	<li>Effective design of PCR bisulphite conversion-specific primers is crucial for ensuring that efficient, unbiased amplification of completely converted DNA occurs. The following guidelines are used to aid primer design.</li>
</ol>
Primer Design Guidelines
<img src="/files/ftp_upload/3170/3170list1.jpg" />
Thermocyling
<ol start="2">
	<li>To test for proportional PCR amplification of methylated and unmethylated DNA, use a 50:50 Methylated/Unmethylated fully bisulphite converted control sample and amplify with the bisulphite conversion-specific primers under the optimized PCR reaction conditions (Figure 3). For the 50:50 Methylated:Unmethylated control, use in vitro SssI methylated DNA and either whole genome amplified (WGA) DNA or whole blood DNA. Please be aware that some genes will be naturally methylated in blood and therefore in these cases it is best to only use WGA DNA as the "Unmethylated" control.</li>
	<li>Prepare PCR amplification reaction mixtures in 100 &mu;l aliquots containing 2 &mu;l of bisulphite converted genomic DNA, 200 &mu;M dNTP's, 1 &mu;M primers, 1.5 mM MgCl2, 50 mM KCl, 10mM Tris-HCL, pH 8.3 and 0.15 &mu;l Taq polymerase.</li>
	<li>In a temperature gradient thermocycler, set the run reaction in a gradient +/- 3&deg;C from the predicted Tm of the primer across 10 tubes</li>
</ol>
<img alt="Table 1" src="/files/ftp_upload/3170/3170table1.jpg" />
<ol start="5">
	<li>To test that the methylated and unmethylated amplicons have been amplified in proportion, the amplicon can be digested with an informative restriction enzyme, such as Taq 1 (TCGA), that will digest methylated DNA but will not digest unmethylated DNA when the restriction enzyme site is altered after bisulphite conversion to TTGA. The extent of digestion can be visualized by agarose gel electrophoresis (Figure 3A). Alternatively, SYBRGreen (0.2 &mu;l of 1:1000 dilution) can be added to the PCR reaction and the extent of methylation can be assessed by performing heat dissociation plots in a real-time PCR thermocycler (Figure 3B).</li>
	<li>PCR Reaction mix including MgCl2 concentration and thermocycling conditions may need to be adjusted to increase PCR efficiency or to ensure proportional PCR amplification of methylated and unmethylated PCR fragments.</li>
	<li>Once the PCR conditions are optimized, PCR amplification can be performed on bisulphite converted samples. Note: Once the Tm is optimized a temperature gradient PCR need not be used.</li>
</ol>
5. Representative Results:
An example of bisulphite PCR amplification optimization is shown in Figure 3. Optimal PCR amplification conditions should amplify methylated and unmethylated amplicons in proportion and without bias. Figure 3A shows an agarose gel with a temperature gradient PCR amplification profile from a mixture of 50% methylated and unmethylated DNA. The PCR amplicons have been treated with a restriction enzyme, Taq 1 (TCGA), that will digest methylated (M) DNA but will not digest unmethylated (U) DNA as the restriction enzyme site is altered by bisulphite conversion to TTGA. An equal amount of cut and uncut PCR product is expected if methylated and unmethylated DNA is amplified in proportion. It can be seen on this gel that the optimal thermocyling conditions for non-bias amplification is at 56.1&deg;C (T). Complete conversion of the bisulphite DNA can also be analyzed by digestion with cytosine-site specific enzyme such as HpaIII (CCGG). HpaIII will only digest if the conversion has failed, as the restriction site will be maintained. If the conversion is complete the restriction site will be converted to TCGG or TTGG depending on the methylation state of the DNA. Complete bisulphite DNA conversion can be seen in Figure 3A (H).
Figure 3B shows a real time dissociation plot that can also be used as a tool to determine whether there is any amplification bias based on the temperature at which the different molecules will dissociate. In this figure it can be seen that the PCR has amplified methylated (M) and unmethylated (U) DNA in proportion from a mix of 50% methylated and unmethylated DNA compared to the control amplification of fully methylated and unmethylated DNA which dissociate at 82.1&mu;C and 78.9&mu;C respectively.
After optimization of thermocyling and reaction conditions Bisulphite treated samples can be amplified with strand specific and bisulphite specific primers in either a single or semi nested PCR reaction. The resulting PCR fragments can be visualized by agarose gel electrophoresis and sequenced either directly (Figure 4A) or by cloning and sequencing. After cloning and sequencing the methylation state of the individual molecules can be tabulated, in a bisulphite map (Figure 4B), to visualize the heterogeneity of methylation.
High throughput quantitative methylation analysis can be preformed using technology such as that utilized by the Sequenom EpiTYPER method. Using this method, bisulphite converted DNA is amplified with bisulphite specific PCR primers and followed by a proprietry cleavage process. The resulting fragments are quantitated by MALDI-TOF mass spectrometry with the specific spectrum dependent on the presence of methylated cytosines (Figure 5A). A summary of methylation ratios in the sample can then be extrapolated in the form of an Epigram (Figure 5B) or methylation plot (Figure 5C).
<img alt="Figure 1" src="/files/ftp_upload/3170/3170fig1.jpg" /> 
Figure 1. Methylated CpG schematic. In the normal cell, promoter-associated CpG islands are predominantly unmethylated (grey) whereas CpG sites within gene bodies are sparse and generally methylated (red). The panel on the right expands the molecular structure of DNA at an individual CpG site and shows methylation with a CH3 molecule at carbon 5 of cytosine.
<img alt="Figure 2" src="/files/ftp_upload/3170/3170fig2.jpg" /> 
Figure 2. Chemical conversion schematic. Analysis of DNA methylation includes four main stages as shown; denaturation, bisulphite conversion, PCR amplification and analysis. In the right panel, modifications to the cytosine molecule that occur during bisulphite conversion are depicted.
<img alt="Figure 3" src="/files/ftp_upload/3170/3170fig3.jpg" /> 
Figure 3. Optimisation of PCR amplification. A. Agarose gel electrophoresis with a temperature gradient PCR amplification profile from a mixture of 50% methylated and unmethylated DNA. Fragments have been digested with either Taq1 (T) or HpaIII (H) which digest methylated DNA and non bisulphite converted DNA respectively. B. Heat dissociation curve analysis shows an equal proportion of methylated and unmethylated DNA (red line 50/50 mix) compared to the control amplification of fully methylated (pink line, M) and unmethylated DNA (green line, U) which dissociate at 82.1&deg;C and 78.9&deg;C respectively.
<img alt="Figure 4a" src="/files/ftp_upload/3170/3170fig4a.jpg" /> 
<img alt="Figure 4b" src="/files/ftp_upload/3170/3170fig4b.jpg" /> 
Figure 4. Example of direct sequencing and a bisulphite map. A. Sequence trace from three different cell lines with CpG sites highlighted in yellow. Cell line X displays 100% methylation at all three CpG sites whereas cell lines Y and Z show varying degrees of methylation as seen by overlapping G/A signals. B. Representative bisulphite map showing the density of methylation (red dots) at individual CpG sites, as determined by direct sequencing of individual clones.
<img alt="Figure 5a" src="/files/ftp_upload/3170/3170fig5a.jpg" /> 
<img alt="Figure 5bc" src="/files/ftp_upload/3170/3170fig5bc.jpg" /> 
Figure 5. High throughput DNA methylation analysis using Sequenom. A. Spectrum view using Sequenom epiTYPER technology. DNA fragments display specific spectra, depending on amount of DNA methylation present. B. Epigram summary of the percentage of DNA methylation at each CpG site for four different cell lines. C. Methylation plot summary derived from the Sequenom Epigram.

<ol>
	<li>Frommer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genomic+sequencing+protocol+that+yields+a+positive+display+of+5-methylcytosine+residues+in+individual+DNA+strands.">A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.</a> Proc. Natl. Acad. Sci. U. S. A. 89, 1827-1831 (1992).</li><li>Clark, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+mapping+of+methylated+cytosines.">High sensitivity mapping of methylated cytosines.</a> Nucleic. Acids. Res. 22, 2990-2997 (1994).</li><li>Grunau, C., Clark, S. J., Rosenthal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisulfite+genomic+sequencing:+systematic+investigation+of+critical+experimental+parameters.">Bisulfite genomic sequencing: systematic investigation of critical experimental parameters.</a> Nucleic. Acids. Res. 29, E65-E65 (2001).</li><li>Clark, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation:+bisulphite+modification+and+analysis.">DNA methylation: bisulphite modification and analysis.</a> Nat. Protoc. 1, 2353-2364 (2006).</li></ol>

No conflicts of interest declared.

DNA methylation analysis by genomic sequencing of bisulphite converted DNA is a well established and versatile method that facilitates the identification and quantification of DNA methylation at single nucleotide resolution. However, bisulphite conversion and subsequent analysis relies on the premise that the DNA has been fully converted with every unmethylated cytosine being deaminated to uracil and only methylated cytosines remaining un-reactive. If conversion is incomplete, problems can arise with analysis since unconverted unmethylated cytosines may be incorrectly interpreted as methylated cytosines. Bisulphite conversion can be optimized at various stages to maximize the percentage of conversion. For DNA to be fully converted it first must be single stranded so that cytosine residues are exposed to the bisulphite ions. The first step, DNA denaturation, is critical and can be the source of incomplete conversion if the DNA is not fully denatured. To ensure that the DNA is fully denatured it is important that the reaction parameters including removal of all associated protein, appropriate salt concentration, incubation temperature and time are suitable to maintain DNA in single stranded conformation. It is important to use fresh 3M NaOH and to allow adequate incubation time. If the DNA is resisting denaturation, some modifications to the protocol including extending the incubation time to 30 minutes or fragmenting the DNA prior to denaturation may facilitate DNA denaturation. In addition, single stranded DNA (ssDNA) may re-anneal to double stranded DNA (dsDNA) during the bisulphite conversion reaction. Carrying out the reaction at a higher temperature (90-95&deg;C) either periodically, or continuously during the bisulphite reaction can aid the maintenance of ssDNA. However, it is important to be aware that DNA degradation is greatly accelerated at these higher temperatures. Various reagents can also be added to disrupt re-annealing of the DNA strands, such as urea.
The concentration of DNA and its quality can also affect the efficiency of the bisulphite reaction and ultimate PCR yield. DNA degradation is a limitation of the bisulphite conversion protocol. The chemical treatment of the DNA introduces various strand breaks in ssDNA and some of the harsh conditions necessary for complete bisulphite conversion including high bisulphite concentration, long incubation times can accelerate DNA degradation. Under the standard reaction conditions described, DNA degradation is not a common problem however, if protocol modifications are introduced, such as for sequences that are refractory to conversion, for DNA templates that are degraded or of poor quality such as FFPE samples, then DNA degradation can become a significant limitation.
Formalin fixed, paraffin embedded (FFPE) and degraded DNA samples can be effectively bisulphite converted and amplified using this protocol, however modifications to ensure that the DNA is not further degraded are advised. The use of a carrier RNA such as tRNA is recommended. Also glycogen can be added as a carrier when DNA concentration is low (&lt;200 ng). Because DNA isolated from FFPE tissues is already fragmented and further fragmentation takes place during deamination, care must be taken to minimize further degradation. Do not fragment DNA any further before denaturation and limit the incubation time for the bisulphite reaction to 4 hours. Design amplicons no larger than 300 bp due to fragmented DNA. 
Another factor that can affect PCR amplification that bisulphite conversion of unmethylated cytosines reduces the complexity of the DNA template. The following primer design protocol outlined in section 4.1, increased primer length and nested PCR can all help to increase specificity and PCR yield. 
Finally, since the DNA template is altered during bisulphite conversion, amplification bias can be a concern. Ensure that proportional PCR amplification of methylated and unmethylated templates is checked with a control 50/50 M/U DNA mix, as outlined in the protocol, see Figure 3. Also ensure that amplification of only converted templates is occurring using HpaIII restriction enzyme and gel electrophoresis (Figure 3). The PCR conditions may need to be optimized by varying the temperature and/or magnesium concentration or lengthening extension time. Some templates appear to be particularly problematic and primer position may need to be adjusted or degenerate primers may need to be used.
Next generation sequencing techniques are rapidly evolving to achieve a similar resolution to bisulphite genomic sequencing, and third generation single molecule sequencing has the potential to allow for analysis of both primary DNA and methylation without the need for bisulphite sequencing. However, widespread availability and specificity of these techniques remain some time away. Bisulphite genomic sequencing is the gold standard in methylation analysis and is a robust protocol. The method has progressed to the point where the common problems and limitations have been resolved and the applications have expanded from individual gene analyses to high throughput and whole genome analysis described by Clark et al. 20064. Troubleshooting guidelines and additional recommendations are outlined in Table 1. It is suggested that the optimum approach is to initially follow the standard protocol and only introduce modification if and when a problem arises.
<img src="/files/ftp_upload/3170/3170table2.jpg" alt="Table 2" /> 
Table 1. Troubleshooting Guidelines

<ol>
 <li>Sodium Metabisulphite (Ajax Finechem) </li>
 <li>Hydroquinone (Merck) </li>
 <li>Wizard DNA-clean-up system (Promega)</li>
</ol>

dna methylation: bisulphite modification and analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

Watch this Scientific Journal Video about DNA Methylation: Bisulphite Modification and Analysis at JoVE.com

DNA Methylation: Bisulphite Modification and Analysis

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

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104.3K Views.  Garvan Institute of Medical Research. Il gold standard per l'analisi di metilazione del DNA è il sequenziamento del DNA genomico di bisolfito convertito. Questo metodo sfrutta l'accresciuta sensibilità della citosina rispetto ai 5-metilcitosina (5-MEC) di bisolfito deaminazione in condizioni acide. Cytosines non metilato può essere distinto dal cytosines metilato dopo amplificazione del DNA genomico di destinazione.

Video: DNA Methylation: Bisulphite Modification and Analysis

PDMS Device Fabrication and Surface Modification

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.