So DNA methylation genome-wide analysis with the LA platform for complex aberration of DNA methylation in the human genome can provide important information of epigenetic biomarkers in gastrointestinal cancer. Though this LA platform for complex aberration of DNA methylation in the human genome, it is optimal in terms of cost and genomic coverage. So demonstrating the procedure will be Hirotaka Momose, a graduate student from my laboratory.
To begin, prepare 10 micrometers of unstained formalin-fixed paraffin embedded sections. Place the slides in a glass slide holder and fill it with saline, making sure that all tissue on the slide is submerged. Leave the slides in xylene for 15 minutes, then pour out the xylene while holding the slides with a pipette tip so that they do not fall out.
Pour in more xylene to the same level as before and repeat the incubation. Pour out the xylene and fill the slide holder with 100%ethanol, making sure that all the tissue on the slide is fully submerged. Leave the slides in the ethanol for three minutes, then replace the ethanol with fresh ethanol and allow them to incubate for two more minutes.
Pour off the ethanol and remove the slides. Carefully place them face up on a clean paper towel and allow them to dry for 10 minutes. Fill a 1.5 milliliter single-use polypropylene tube with 80 microliters of lysis buffer and put a clean pipette tip into the buffer.
Identify cancer tissues of the tumor area most suitable for macrodissection according to the appropriate H&E stained section, then macrodissect the cancer tissue based on the marked section. Take a clean razor blade and gently scrape the cancer tissue off of the slide trying to keep it in one piece. Use the pipette tip to transfer the scraped tissue into the lysis buffer vial.
Repeat this process with the rest of the slides. After all the tissue is in the tube, use the tip to make sure the tissue was fully submerged and is not stuck to the wall. Add 20 microliters of a subtilisin-related serine protease to the vial and gently flick to mix.
Place the vial in a 55 degree Celsius heat block for at least four hours or overnight, making sure to slightly vortex after two hours. Perform the bisulfite treatment on 45 microliters of the digested tissue using a bisulfite conversion kit according to the manufacturer's instructions. Add five microliters of dilution buffer to the sample and incubate it at 37 degrees Celsius for 15 minutes.
Meanwhile, prepare the bisulfite conversion reagent by adding 750 microliters of distilled water and 210 microliters of dilution buffer to one tube of CT conversion reagent. Mix the tubes by vortexing for 10 minutes, then add 100 microliters of the prepared CT conversion reagent to each sample and mix by inversion. Incubate the samples in the dark at 50 degrees Celsius for 12 to 16 hours.
After the incubation, place the samples on ice for 10 minutes. Add 400 microliters of binding buffer and mix each sample by pipetting up and down. Load each sample into a spin column and place the column into a two milliliter collection tube.
Centrifuge the samples at full speed for one minute and discard the flow-through. Add 200 microliters of wash buffer to each column and spin at full speed for one minute, then discard the flow-through. Add 200 microliters of desulphonation buffer to each column and allow the columns to stand at room temperature for 15 minutes.
After the incubation, spin the columns at full speed for one minute and discard the flow-through. Wash the column twice with 200 microliters of wash buffer centrifuging for one minute at full speed after each wash. Add 46 microliters of distilled water to each column and place it in a new sterile 1.5 milliliter single-use polypropylene tube.
Spin the tubes for two minutes to elute the DNA and discard the column. The DNA is now ready for the analysis. Use the bisulfite modified DNA as a template for quantitative methylation-specific PCR to evaluate methylation of the promoter region in each gene analysis.
Combine the reagents as described in the text manuscript and use a 96-well real-time PCR instrument to run the thermo cycling protocol. The characteristics of 48 patients with gastric cancer in the training cohort are shown here. The median age of patients was 74 years and the cohort included 38 males and 10 females.
First, all 48 samples were loaded for identification of outliers. Two samples gave peaks that were greater than two standard deviations displaced from the others and these samples were removed. The resultant heat map was divided into two groups based on high and low methylation.
This heat map allows visualization of the top 50 probes within 1, 500 base pairs of the transcriptional start site in the differential methylation analysis. The type of cancer and presence of lymph node metastasis emerged as significant independent predictive factors when the clinical pathological factors were used as covariates in the multivariate analysis. Finally, the EPB41L3 gene was found to be strongly associated with codifying the training cohort into high and low methylation groups in the microarray analysis.
The results of the microarray analysis for EPB41L3 in the test cohort were validated with quantitative methylation-specific PCR. RMVs of EPB41L3 in PGC tissues were significantly higher than those in remnant gastric cancer in univariate analysis. Similarly, RMVs in samples with lymph node metastasis were significantly higher than those without lymph node metastasis.
So identification on cancer tissue most suitable for macrodissection according to the appropriate H&E stained section is required to successfully perform this protocol.
