Macrodissection of FFPE tissue prior to nucleic acid purification allows the operator to select tissue of interest (typically tumor tissue) and minimize background tissue. This in turn can enrich the extracted nucleic acid for variants of interest.
The Ionic Purification System from Purigen Biosystems is an excellent choice for macrodissected tissue. It offers a simple, automated work-flow and typically generates yields that are 2-4x higher than other methods, maximizing the amount of nucleic acids extracted from macrodissected tissue.
Macrodissection consists of 3 primary steps: marking, scraping, and transferring for downstream analysis. Care must be taken at each step to ensure the desired tissue is safely captured and unwanted tissue is appropriately disposed.
Macrodissection starts by defining an area of interest. Typically, a pathologist will mark one stained slide (e.g. with H&E). The markings can then be transferred to the unstained slides to be used for nucleic acid collection.
The precision of the marking process can impact the degree of enrichment in the nucleic acid preparation. To get the most precise area selection:
In some cases, it is desirable to simply remove paraffin from the sample and collect only the tissue. In these cases, it may be possible to mark directly on the unstained slide, without using a stained section.
FIGURE 1: Place a slide with unstained tissue on top of a slide with stained tissue and mark the desired area on the unstained slide. Both slides should be oriented with the FFPE slice on the bottom and the area of FFPE between the two slides should be aligned.
Once the desired tissue section is marked, the next steps are to trim the waste material, then collect the desired material. Both steps are typically performed by hand with a sharp, disposable blade.
Safety is of critical importance at this stage because there are two sharps hazards in play during the operation – the blade, and the potential for a broken glass slide.
Start by using appropriate PPE: safety glasses, disposable gloves, and a lab coat. Make sure that each element fits properly and does not obstruct movement.
Use the appropriate technique during scraping:
We recommend practicing on non-critical FFPE sections before performing critical work.
Once the technique is clear, proceed to the macrodissection. First, scrape away the tissue not desired for the purification, leaving only the desired tissue. Then, come back and collect the desired region.
FIGURE 2: Carefully scrape away undesired material and discard in an appropriate disposal receptacle.
Transfer for Downstream Analysis
This scraping of the desired tissue is the most important step. It is ideal to scrape it in a single pass if possible, because this results in the most compact piece of tissue to transfer into the tube. However, this is not always possible, and will depend strongly on the tissue in the block and thickness of the section.
Transferring the collected tissue to a tube can be a challenge for several reasons.
A couple of techniques help mitigate the challenge.
These operations usually still result in tissue stuck to the side of the tube. One way to encourage them into the bottom of the tube is to centrifuge the tube. Centrifugation at maximum speed for 2 minutes is usually sufficient to move most of the material to the bottom.
It is critically important that the tube you transfer to at this stage is the tube intended for lysis. Any additional transfer after this stage is likely to result in lost tissue.
FIGURE 3: Create a “scroll” with the desired tissue by scraping in a smooth motion that curls the tissue section around itself. Ideally this is done in a single scrape. Use a pipette tip to transfer the scroll to an Eppendorf tube.
Select the appropriate Ionic Purification Kit depending on your needs and follow the protocol provided with the kit: