How can you increase your chances of success with FFPE?
Although the Optimum FFPE Isolation Kit is a robust protocol, there are other uncontrollable factors that can impact the overall quality and yield of RNA isolated from FFPE tissues. Below are a few recommendations to address several key factors.
1) Upstream Tissue Procurement and Tissue Specimen Preparation If possible, tissues should be fixed within one hour of surgical resection. Extensive degradation of RNA can occur before completion of the fixation process. The optimal fixation time is 12-24 hours, using neutral-buffered formalin or paraformaldehyde. Fixed tissues should be thoroughly dehydrated prior to embedding.
2) Tissue Type, Size, and Amount Being Used for RNA Isolation The recommended tissue thickness is 10-20 m (Table 1). The number of sections used is determined by the tissue type (which impacts cell density) and surface area (recommended size: 50-300 mm2). Excess starting material can cause filter clogging, resulting in poor yield.
3) Excessive Amount of Paraffin Used for Embedding
Tissues Two times xylene treatment at room temperature should
be sufficient for complete deparaffinization. If desired, a more rigorous
37-55C treatment can be done for up to 30 minutes. After the xylene deparaffinization,
it is crucial that the 100% ethanol is completely removed and the pellets