Optimum FFPE RNA Isolation Kit offers a simple and rapid protocol for phenol-free isolation of total RNA from formalin or paraformaldehyde fixed paraffin embedded (FFPE) tissues that is optimized for RT-PCR analysis. The kit is designed for use with small amounts of sample material, and the Micro Filter Cartridge allows RNA to be eluted in a concentrated form. The kit is also compatible with cells from LCM samples.
Why use FFPE tissues?
FFPE tissues are the most widely available specimens for retrospective clinical studies of disease mechanisms. These archived materials provide a valuable source of stable RNA for gene expression analysis, using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). As the use of PCR technology has become more prevalent in molecular testing, it has enhanced the clinical utility of FFPE tissues; e.g. retrospective analysis of archival tissue would enable the correlation of molecular findings with the response to treatment and the clinical outcome.
Why is RNA isolation from FFPE such a challenge?
The recovery of quality RNA from FFPE specimens can be quite challenging. 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, respectively.
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 are dry after the 2X 100% ethanol wash .
Table 1. The approximate range in RNA yield from human kidney FFPE sections of varying thickness.
Proteinase K Solution and Buffer
RNA Extraction Solution
Micro Filter Cartridge/ Waste Collection Tube Assembly
RNA Elution Solution and Tubes
RNase-Free DNase I and Buffer
DNase Inactivation Reagent
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