Patho Entrance Biomarkers

Why do we have to perform deparaffinisation step during nucleic acid isolation from FFPA (formalin fixed paraffin tissue) tissues?

because paraffin wax, being hydrophobic and water-repellent, hinders the isolation of genetic material. The chemicals used in the isolation process are water-based and cannot effectively penetrate or interact with paraffin. Failure to remove the wax would impede the extraction of DNA or RNA.

Why do we have to perform high temperature incubation at 70 degrees during the isolation of nucleic acids from FFPA tissues?

Formalin fixation introduces cross-links between proteins and nucleic acids, making it challenging to extract DNA from the nucleus. The 70-degree incubation serves to reverse these formalin-induced cross-links, facilitating the extraction of DNA and enabling successful isolation.

Why do we have to add lysis buffer to isolate nucleic acids?

It is a detergent which breaks down cellular and nuclear membrane to release the DNA from the nucleus.

Why do we add proteinase during DNA/ RNA isolation?

To be able to digest or degrade the proteins

Why should we remove proteins during DNA isolation?

TO OBTAIN CHEMICALLY PURE DNA

Chromatin-between histones and DNA. In DNA isolation, we have to obtain pure DNA. If in the nucleus, the DNA is bound to histones, we have to remove the histones, and other proteins in the cell.

Why do we have to properly store and preserve patient samples collected for molecular biology studies?

It is crucial to prevent DNA and RNA degradation. The presence of ubiquitous enzymes, such as DNAases and RNAases, necessitates correct storage to minimize their activity. The quality, quantity, and integrity of isolated nucleic acids directly impact downstream procedures, emphasizing the importance of meticulous sample preservation.

How do we correctly store and preserve patient samples collected for molecular biology studies?

1. Freezing:

   • -70°C (dry ice): Short-term storage.

   • -80°C (ultra-low freezers): Long-term storage.

   • Purpose: Decreases nuclease activity.

2. Liquid Nitrogen:

   • Storage at -196°C: ultra-low temperature environment for preserving nucleic acids.

3. Commercial Solutions:

   • Inhibitors: Prevent RNAase and DNAase activity.

Give 4 examples of molecular biology techniques used is diagnostic pathology?

FISH, PCR, RT-PCR, NGS (next generation sequencing)