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

The overall goal of this procedure is to assess the DNA methylation status of KCNJ 10 in an enriched population of astrocytes and correlate DNA methylation changes of the promoter with transcriptional optional activity. This is accomplished to by first isolating an enriched population of cortical astrocytes from whole rodent brain via facts sorting next DNA is isolated from the enriched population of astrocytes. Then the DNA methylation status of KC J 10 is assessed by using methylation sensitive high resolution melt analysis or MS.HRMA.

Finally, the KC J 10 promoter is hypermethylated and lucifers assay is utilized to correlate changes in DNA methylation of the promoter with transcriptional activity. Ultimately, MS.HRMA and the luciferase assay are used to measure the DNA methylation status of KCNJ 10 and correlate changes in the methylation of the promoter and transcriptional activity of the gene Demonstrating the procedure will be seen. A postdoctoral fellow from my laboratory, All animals were handled in accordance with the National Institutes of Health Guidelines, the Animal Care and Use Committee at the University of Alabama at Birmingham approved animal use.

All buffers and reagents were prepared according to the text protocol. After dissecting the cortices from the head of a euthanized animal, according to the text protocol, cut or dremel a hole in the top of a 50 milliliter conical tube to allow tubing to be fed into the tube. Then add papin solution to the tube.

Place the dissected cortices in a 10 millimeter culture dish containing dissociation medium equilibrated to 95%O2 to 5%CO2, and use a clean razor blade to mince the tissue into one by one square millimeter pieces. With a 10 milliliter pipette, men transfer the tissue to the 50 milliliter tube containing the pepane solution. Allow the tissue to settle to the bottom of the transfer pipette before discharging.

To minimize the amount of dissociation medium carried over without bubbling the pepane solution, apply a constant flow of 95%O2 to 5%CO2 via surface gas exchange while incubating in a 37 degrees Celsius water bath for 20 minutes. After the incubation, use a 10 milliliter transfer pipette to slowly tate the tissue 10 times centrifuge the cloudy cell suspension at 300 G for five minutes at room temperature. Equilibrate the DNA's albumin inhibitor solution via surface gas exchange and resuspend the pelleted cells in three milliliters of the solution.

Then prepare a commercial discontinuous density gradient following the manufacturer's instructions. Spin the discontinuous density gradient at 70 G for six minutes. Then isolate the dissociated cells from the bottom of the tube by using a pipette to aspirate off media.

Use two to three milliliters of DPBS with 0.02%bovine serum albumin and one milligram per milliliter of DNA or preferred media. To reus, suspend the dissociated cells. Pass the cells through a 40 microgram filter before carrying out fact sorting and DNA or RNA extraction according to the text protocol.

After designing primers against a bi sulfite converted DNA sequence and amplifying the DNA according to the text protocol by sulfite convert 500 to 1000 nanograms of DNA of each sample and methylated DNA standards ranging from zero to 100%from the same animal species using a preferred DNA polymerase and five micromolar primers. Set up 20 microliter reactions for Ms HRM amplification. According to this table, run all the samples including fax, DNA and methylated standards in triplicate depending on analysis software set, pre and post start and stop parameters around the transitions of the melt curve.

Set the prem melt, start and prem melt stop parameters. So the difference between the two is 0.2 to 0.5 degrees Celsius. Set post melt, start and stop similarly and extract peak temperature difference data for each sample using percent methylated standards and their corresponding peak temperature differences.

Generate a linear regression equation. Use this linear regression equation to estimate methylation status of unknown samples after identifying CPG islands of interest and amplifying and cloning CPG island Luke two plasmid according to the text protocol. Use preferred cutter software to verify sites for restriction digest to avoid double cuts and linearize 30 micrograms of plasmid once the sample has been digested with the appropriate enzymes.

Heat inactivate enzymes at the appropriate temperature and duration in 50 microliter reactions. Use five units of CPG methylate to methylate. 700 micrograms of linearized plasmids at 30 degrees Celsius for 13 to 19 hours.

After cleaning the DNA as outlined in the text protocol, carry out an HPA two restriction digest on one microgram samples of methylated DNA by incubating at 37 degrees Celsius for one hour. After cleaning the DNA run samples on a 1%agros, DNA gel at 100 volts for 45 minutes for visualization. Next subject, methylated and non methylated plasmids to double digestion with appropriate restriction enzymes overnight to release the full length Luke two vector and CPG island fragments.

Heat inactivate the enzymes following digestion. Run the double digested samples on a 1%DNA agros gel at 100 volts for one hour to separate vector and insert then while wearing protection against UV light, place the gel on a black light and with clean surgical blades, excise the methylated and non methylated inserts after gel extracting the DNA according to the text protocol, use T four DNA Ligase and a one to four ratio of vector to insert to relegate the methylated and non methylated inserts into a unmethylated vector. Incubate at negative 20 degrees Celsius or on ice overnight following the ligation.

Clean the DNA and verify re ligation by running samples on a 1%DNA aros gel and assess the concentration of re ligated plasmid CD 54 cells on a 12 well plate at 140, 000 cells per well. 24 hours later. Use a commercial transfection reagent to transfect cells with equal concentrations of either non methylated CPG loop two plasmid plus RAN vanilla or other control ferous vector or methylated c pg Luke two plasmid plus ELLA or other control lucifers vector Allow the cells to transfect for 24 hours before performing a lucifers assay.

According to the manufacturer's instructions, use luminometer to read each well in triplicate. Calculate the ratio of firefly lucifers activity to ran vanilla or other control. Lucifers activity.

Normalize the methylated firefly control luciferase activity to non methylated firefly control luciferase activity by dividing methylated activity by methylated luc activity. As demonstrated here, an enriched population of astrocytes was acquired via fax sorting of E-G-F-P-S 100 beta transgenic animals. A gated population was targeted based on forward and side scatter, and a live cell population was determined using an bromide dead cell indicator and gating shown here are representative images of EGFP positive astrocytes following dissociation, but prior to sorting as well as after sorting, this figure shows an isolated EGFP positive cell population that demonstrated a 40 fold increase in mRNA for the astrocyte specific marker.

A LDH one L one. In addition, despite shared expression of S 100 beta and NG two positive OPCs and astrocytes, a fourfold reduction in NG two mRNA, A marker for oligodendrocyte precursor cells was observed indicating isolation of an enriched astrocytic cell population. This table lists total RNA and DNA, isolated from varying ages and number of animals, and is listed as a reference for expected yield of molecular molecules following facts.

Finally, a dual lucifers assay was utilized to assess the transcriptional activity of hypermethylated regions of the gene of interest after moving the methylated insert into a non methylated vector. HPA two, which digests only non methylated DNA was used to verify the methylation status of the plasmid. After watching this video, you should have a good understanding of how to isolate an enrich population of astrocytes using facts as well as how to measure DNA methylation of a gene and correlate changes in the DNA methylation of the promoter with transcriptional activity using methylation sensitive high resolution melt analysis and luciferase assay respectively.