The overall goal of this procedure is to study DNA. Methylation and gene expression changes upon early life stress. This is accomplished by first subjecting newborn mouse pups to maternal separation to induce early life stress as a second step brain areas of interest here, the PVN are obtained by in loco micro dissection and DNA and RNA are isolated simultaneously from a single tissue punch.
Next, the obtained DNA is by sulfite treated, and the region of interest is amplified by bi sulfite PCR. This is then ligated into a vector and transformed into bacteria. Successful transformations are identified by marker expression and PCR fragment size.
And lastly, bi sulfide sequencing is used to assess methylation status. The main advantage of this workflow is that it allows convenient analysis of epigenetic programming in response to environmental stimuli. This method can provide insight into epigenetic programming by early life adversity.
It can be also applic to other systems. Wherever methylation and gene expression are analyzed in highly tissue specific environment and wherever tissue availability is limited To induce early life stress. Maternal separation is performed on pups from timed pregnant C 57 black six N dams to affect maternal separation.
Transfer each litter to a heated clean cage for three hours daily from postnatal days one to 10 leave unstressed control pups undisturbed. Apart from the three hour separation, all litters are left with their mothers until weaning on postnatal day 21. Then the pups are housed in sex matched groups, three to five mice per cage under standard housing conditions to initiate the collection of brain tissue, the mouse brains are removed from the skulls and immediately sna frozen in isop dry ice and stored at minus 80 degrees Celsius.
When ready to collect the tissue punches, remove the brains from the minus 80 degree freezer and put them on dry ice. Begin by cryosectioning the brains into 10 micron coronal sections from rostral to coddle. Mount the sections on super frosts glass slides and dry them for crest violet staining.
Additional slides can be taken and stored at minus 20 degrees for further analyses, such as in situ two hybridization. When ready to take punches, stain the slides with crestal violet to identify brain structures of interest. Then using a punching needle, take 0.8 millimeter punches of the regions of interest using in loco microdissection.
The punches should be stored at minus 80 degrees Celsius. It's of critical importance that micro punching is performed in standardized manner as gene expression and methylation are highly tissue specific. Homogenize the punches in 400 microliters of qadium thiocyanate buffer using a pipette then vortex at room temperature.
Next, pass the resultant lysate through a 29 gauge syringe. Several times the punch should no longer be visible. Split the lysate into equal parts.
One to process RNA, which should be done first and the other to process DNA, which may be stored at room temperature until the RNA is processed for RNA purification at a one 10th volume of sodium acetate, one volume of aqua phenol and a half volume of 24 to one chloroform to isoamyl AML vortex. The mixture vigorously after each addition and incubate the final concoction on ice for 10 minutes. Then centrifuge the mixture.
Collect the aqueous phase and add an equal volume of 70%ethanol to it. Using an RNA spin column kit. Perform an on column DNA's digestion and wash elute the RNA in 25 microliters of water.
Now purify the DNA using a kit protocol modified to include the addition of an RN a's digestion, and to warm the elucian buffer and elucian column to 70 degrees Celsius before their use. 10 minutes at 70 degrees Celsius suffices for the columns. Finally, use a spectrophotometer to determine the DNA and RNA concentrations.
A typical PVN punch yields about 600 nanograms of DNA and about 400 nanograms of RA set aside, about 100 nanograms of RNA for gene expression analysis by quantitative PCR prior to amplification by sulfite. Treat 200 nanograms of the isolated DNA with a commercially available kit to amplify DNA from the methylated regions. Order primers designed specifically for bi sulfite converted DNA Optimal primer design in bi PCR is essential as the quality and the specificity of the PCR amplicon determines the success of the following steps.
When the primers arrive, determine their optimal and kneeling temperature with pilot experiments. Use two microliters of bis sulfite treated DNA as a template for each 25 microliter. PCR mixture.
Amplify the mixture starting with a six minute denaturing at 95 degrees Celsius, followed by 45 to 50 amplification cycles before a final five minute elongation at 72 degrees Celsius. Analyze seven microliters of the PCR product by Agros gel electrophoresis to verify size of the amplicon. Purify the remaining PCR product for subsequent ligation using a commercially available PCR cleanup kit.
If undesired PCR products are obtained, use gel purification to isolate the product of interest for this protocol. A commercial cloning kit is employed. Cloning efficiency depends critically on the insert.
So if low numbers of recombinant clones are repeatedly obtained, try a different cloning vector. Set up a 10 microliter ligation reaction with one microliter of vector and three microliters of cleaned up PCR product. Mix the reaction by pipetting and incubate it overnight at four degrees Celsius the next day.
Clean up the ligation reaction by classical ethanol precipitation and transform the bacteria with the product. Add one milliliter of prewarm SOB medium directly after the pulse delivery and transfer the transformed bacteria into a reaction tube. After recovering bacteria for one hour at 37 degrees Celsius, spread 100 microliters of each suspension on an LB ampicillin plate coated with IPTG.
Xal incubate the plates overnight once colonies can be picked. Set up 25 microliter colony PCR reactions in a 96 well plate using three microliters of 2.5 millimolar magnesium chloride in each reaction. Pick positive white clones with a pipette tip and transfer them to a PCR mix with a simple dip.
Begin the PCR with a four minute melting cycle. Follow this with 10 amplification cycles with an ling temperature of 56 degrees Celsius. Each step of these cycles is 30 seconds.
Then decrease the ealing temperature to 48 degrees Celsius for 30 more amplification cycles. Finish with a five minute elongation cycle. Now load five microliters of each product on an aros gel and identify reactions that contain the right sized insert.
Use a commercially available kit to clean up the PCR products of interest so that they can be sequenced using big dye terminator cycling sequencing. In a 96 well plate load wells with three microliters of big DI reaction master mix, followed by two microliters of cleaned up colony PCR product. Run the reaction on a thermocycler beginning with one minute at 96 degrees Celsius, followed by 35 cycles of 10 seconds at 96 degrees Celsius, five seconds at 50 degrees Celsius, and four minutes at 60 degrees Celsius.
After the big die reaction is finished, clean up the reaction using a big die purification plate. After obtaining sequences, analyze them using the online big analyzer or B-I-S-M-A tool to derive the methylation pattern of the investigated DNA region to gain insight into the influence of early life stress or ELS on a VP expression and methylation status. C 57 black sticks and mice were processed according to the protocol described, the paraventricular nucleus and supraoptic nucleus were punched to isolate DNA and R-N-A-D-N-A was bi sulfite treated, amplified with primers specific to the a VP enhancer and the PCR products were cloned and sequenced at least 20 recombinant clones from each mouse.
PCR were analyzed to determine the methylation frequencies for the CPGs contained in the PCR amplicon in tissue from the P-V-N-E-L-S induced a significant hypomethylation at four locations on the A VP enhancer suggesting epigenetic marking of this regulatory region through early life experiences. In contrast to the PVN methylation of the A VP enhancer was unaffected by ELS in the SON illustrating tissue specificity of the epigenetic effect in control animals, DNA methylation status at CPG 10 correlated negatively with a VP gene expression pointing to a role of DNA methylation in the fine tuning of a VP gene expression. Following this procedure, other methods like UPCR can be readily performed on the isolated RNA in order to answer additional questions like gene expression changes in the analyzed tissue.
After watching this video, you should have a good understanding of how to study DNA methylation changes in response to environmental or experienced dependent stimuli.
