Optimized Storage Methods of RNA Extraction from Formalin Fixed Paraffin Embedded Tissue

Abstract

Background: RNA extraction from Formalin Fixed Paraffin Embedded tissue (FFPE) provides valuable information. The main obstacle for pure RNA extraction from FFPE specimens is RNA degradation over time and low yield of RNA due to chemical processing. In the present study, RNA extraction from FFPE specimens were optimized for storage time, proteinase K concentration, and tissue size hemogenation. Methods: To evaluate the effect of storage time on RNA extraction yield, total RNA from 78 FFPE breast tissue specimens (1 - 3 years n = 52, and less than one year, n = 26) were extracted by High Pure Paraffin Kit (Roche). The effect of 2 different proteinase K on RNA was evaluated by proteinase K, and the effect of homogenization was evaluated using 2 different section sizes (10 µm and 5X2 µm). Extracted RNA was converted to cDNA. The SYBER Green Real time PCR was performed for quantitative analysis using ABI7900 Real time PCR. Results: The results indicated that FFPE storage time affected the yield of RNA extraction. The more the time of storage, the less RNA could be obtained (r = -0.38, P = 0.01). Smaller tissue section size seems to increase the amount of efficient RNA extraction from FFPE, probably through appropriate tissue lysis and more RNA release. According to the current study, proteinase K (Endopeptidase K) from different companies also affected on the quality of RNA extracted from FFPE (P = 0.032). Conclusion: Different optimization strategies enhance quality, purity, and quantity of RNA extracted from FFPE, which is critical in gene expression studies, like qRT-PCR.