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Optimum FFPE RNA Isolation Kit

ng. The fixation process causes cross-linkage between nucleic acids and proteins, and covalently modifies RNA by the addition of monomethyol groups to the bases. As a result, the molecules are rigid and susceptible to mechanical shearing, and the cross-links may compromise the use of RNA as a substrate for reverse transcription. Therefore, in order to utilize FFPE tissues as a source for gene expression analysis, a reliable method is required for extraction of RNA from the cross-linked matrix.


How can you evaluate RNA quality by qRT-PCR analysis?
Ambion Diagnostics' Optimum FFPE RNA Isolation Kit has been optimized for qRT-PCR on fixed embedded tissues. Figure 1 shows that the qRT-PCR results from FFPE and corresponding frozen human kidney tissues are comparable. The generally acceptable range is approximately a 34 Ct difference.

Note: The relatively high Ct value for the frozen tissue indicates that the RNA quality of the original tissue had already been compromised prior to fixation.

Figure 1. qRT-PCR Analysis of GAPDH mRNA in Matching Frozen and FFPE Human Kidney Tissues. The kidney tissue sample was divided into two sections. One section was frozen and embedded in OCT. The other section was fixed in formalin for 24 hours and embedded in paraffin. Two, 10 m sections were used for RNA isolation. RNA was isolated using the RNAqueous Micro Kit for the frozen tissue and Optimum FFPE RNA Isolation Kit for FFPE tissue,
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