FFPET or (Formalin-fixed paraffin-embedded tissue) can be considered an invaluable asset in the world of genetic research. The importance and utility of tissue cores in a paraffin block are indispensable, to say the least. That being said, the overall yield of nucleic acid during is low due to the complicated composition of heterogeneous tissues.
Why it is important to prepare tissue samples in paraffin blocks?
Genetic research that carried out surrounding genomic biomarkers has one and only one aim which is to identify molecular correlations. Identification of molecular correlations enables correct and reliable diagnosis as well as prognosis of both diseases and their treatments respectively.
Preparing biomarkers helps researchers to carry out a retrospective analysis of tissue samples. It is important to store both normal and diseased tissue samples in a frozen state in biobanks. One can also store them as FFPET blocks in medical archives.
Let us now take a look at the preparatory process for paraffin embedded tissue samples:
- Tissue Coring – It is recommended to outline the region of interest with a fine point marker in the microscope slide. Cut a section of paraffin film large enough to cover the region of interest on the slide.
- Deparaffinized Tissue Cores is a must – The process must take place in a 2 ml tube followed by addition of around 1 ml xylene. Vortex the tube for around ten seconds and heat at 50 degrees for three minutes. At the end of it all, centrifuge the tube for two minutes at 25 degrees (RT).
- Homogenization of Cores – It is recommended to resuspend the cores in hundred percent pure alcohol before they are being homogenised.
- Digestion with Proteinase K – After homogenization, it is recommended to resuspend the tissue cores in around a hundred and fifty µl of Proteinase K. It is a digestion buffer that loses up the pellet. Post addition of the Proteinase K, it is best to incubate the contents at around 56 degree Celsius for 15 minutes all the while mildly agitating the tube.
- Measures to take during separation of RNA from DNA – Be careful while you are performing this step. It is best to keep the pellet undisturbed when you are transferring the supernatant. When it comes to storing the pellet, you can store the same at room temperature for about two hours to twenty-four hours at two to eight degree Celsius. In case, you want to store the pellet for more than one day, it is best to keep them stored at negative 20 degrees so that the genetic material is intact.
Biomarkers play an important part in genetic research especially when it comes to the treatment, diagnosis and prognosis of cancer. Similar to other types of diseased tissues, a cancer tissue also shows a lot of heterogeneity that is confined to a particular region both in terms of cell type and preservation. Biomarker heavily relies on the correlation of constituents of diseased tissue with the molecular features of healthy tissue. It is one of the many reasons why researchers should take careful steps to make sure that the samples are prepared by following the steps mentioned above.