The overall goal of this procedure is to sequentially extract RNA and DNA from archival formalin-fixed paraffin-embedded tissue cores. This is a protocol for harvesting tissue cores from which we will extract both RNA and DNA. The main advantage of this protocol is that it improves RNA and DNA yields from precisely-targeted regions in the tissue block.
This procedure is largely developed in archival prostate cancer tissues. It can also be applied to other types of archival tissues. The visual demonstration of this method is critical as the tissue coring steps are not commonly used in research laboratories.
Most research labs use cross sections instead, which exhausts tissues faster and adds significant heterogeneity to the sample. Begin by reviewing the microscope slide and outlining the region of interest using a fine point permanent marker. Next, cut a section of paraffin film large enough to cover the region of interest on the microscope slide.
Then, place the film firmly onto the slide, wrapping the film over the edges to keep it from slipping. Using a fine point permanent marker, outline the entire tissue and the region of interest within the tissue. Next, remove the film from the slide and transfer it to the corresponding tissue block.
Orient the film by flipping or rotating it so that the outline of the entire tissue matches the observed shape of the tissue in the block. Press the section of film firmly to the surface of the block to prevent slippage. Using the tip of the permanent marker, make shallow but visible indentations along the outline of the region of interest and then remove the film.
Next, clean the receptor punch from the 0.6 millimeter punch set by submerging the tip into 1.5 milliliter micro centrifuge tube containing one milliliter of bleach and sliding the punch up and down several times. Repeat the wash with the tube containing 70 percent ethanol, and then water. Now press the cleaned punch into the region of interest to a depth of three millimeters and withdraw.
Release the core into a low-binding micro centrifuge tube by pushing it out of the punch with the stylus. Add one milliliter of xylene to the microfuge tubes containing the tissue cores and vortex vigorously for ten seconds. Then place in a heat block set to 50 degrees Celsius for three minutes.
Following the incubation, centrifuge for two minutes at room temperature at maximum speed. After the centrifugation, place the cores on ice for five minutes to solidify the waxy residue. Now, carefully remove the paraffin that has accumulated around the meniscus along with the supernatant using a pipette tip.
Then, repeat the xylene treatment. After removing the paraffin xylene mix, add one milliliter of 100 percent ethanol and vortex vigorously for ten seconds. After centrifuging at maximum speed for two minutes, carefully discard the ethanol.
Repeat the ethanol wash once more before beginning the homogenization. Resuspend the deparaffinized cores in 700 microliters of 100 percent ethanol prior to homogenization. Use a motorized tissue homogenizer on a medium setting to grind the cores into fine particles.
Thorough homogenization of the tissue cores is required for optimum DNA and RNA yield. Next, fill separate 15 milliliter tubes with approximately ten milliliters of bleach, RNase neutralizing solution, and 70 percent ethanol. After sample homogenization, wash the homogenizer probe in each of the cleaning solutions in order.
Run the homogenizer on the highest speed during the washing stage. Wipe the probe with a tissue and allow the probe to dry completely before homogenizing the next sample. Visually inspect the probe blades for residual tissue pieces and if found, clean the probe again.
Following homogenization, bring the sample volume to one milliliter with 100 percent ethanol, then centrifuge at maximum speed for 15 minutes. Carefully aspirate the ethanol and air dry the pellets for approximately 15 to 20 minutes before beginning proteinase K extraction. Resuspend the pellets in 150 microliters of proteinase K digestion buffer and flick the tubes to loosen the pellet.
Overnight proteinase K digestion is recommended for higher DNA recovery. Add 10 microliters of temperature-stable proteinase K and mix by flicking. Incubate at 56 degrees Celsius for 15 minutes with mild agitation.
Incubate the tubes on ice for three minutes. After cooling, centrifuge the tube for 15 minutes at maximum speed. Next, without disturbing the pellets, carefully transfer each supernatant to a new micro centrifuge tube for RNA purification.
Extract RNA and DNA using the optimized procedure in the written protocol, which includes an extended tissue digestion time for DNA extraction. RNA and DNA can be recovered from older formalin-fixed paraffin-embedded tissue samples using this method. Nucleic acids were coextracted from prostate cancer samples ranging from three to 14 years in sample age.
The average yield was 2, 270 nanograms of RNA and 820 nanograms of DNA. Interestingly, there was no significant correlation between the age of the tissue sample and nucleic acid recovery. Overall, RNA and DNA yields were correlated across samples, although in most cases, more than twice as much RNA was recovered relative to DNA.
To demonstrate the performance of genomic DNA extracted with this protocol, bisulfite converted DNA extracts from archival prostate cancer samples were amplified by methylation specific PCR. ALU repetitive elements were used as a methylation control and showed little variation between samples. GSTP1, a gene known to be hyper-methylated in prostate cancer, showed no detectable amplification by methylation specific PCR when DNA extracted from benign samples was used.
DNA samples from patients with aggressive prostate cancer exhibited detectable QPCR cycle threshold values that were lower than those from indolent prostate cancer cells, indicating increased methylation of GSTP1 in aggressive prostate cancer. Once mastered, this technique can be done within three to four hours for RNA, and following overnight digestion, in four hours for DNA if it is performed properly. This technique should enable researchers in molecular pathology to explore diagnostic DNA and RNA biomarkers in a variety of cancers.
After watching this video, you should have a good understanding of how to prepare tissue cores for extraction of DNA and RNA from precisely mapped areas of interest in archival tissue blocks.
