RNA from formalin fixed and paraffin embedded material is chemically modified and cross-linked with other nucleic acids and with proteins preventing efficient isolation, and many fundamental experiments in the field of molecular biology are inhibited or very inefficient. Before RNA can be used efficiently for quantitative and qualitative analyses, e.g. reverse transcription followed by PCR, quantitative PCR and synthesis of probes for DNA microarrays, these modifications must be removed. Such a process is called demodification.
Several published reports describe methods to isolate RNA from fresh (or quick frozen) and from formalin fixed, paraffin embedded (FFPE) cells or tissues. Most of these techniques utilize a cell or tissue disruption step in which the tissue is dispersed in a powerful protein denaturation solution containing a chaotropic agent (e.g. guanidinium or lithium salt). This rapid disruption of cell membranes and inactivation of endogenous ribonuclease is critical to prevent degradation of RNA during purification and isolation.
Generally, RNA is used to gain information on the expression of genes in tissue samples. Methods are often based on quantitative reverse transcription-polymerase chain reaction (qRT-PCR), probably the most sensitive and reliable assay available for quantification of RNA. The qRT-PCR method tolerates fragmentation of starting RNA to some degree and protocols are available to make the measurement robust and reproducible. Another important technological application is microarray expression profiling which is another method based on RNA which also involves a reverse transcription step. It is distinct from qRT-PCR as so far it depends on high quality RNA and special methods are required which tolerate fragmentation of RNA.
RNA is a particularly labile molecule, it is susceptible to nonspecific degradation by physical conditions, mainly heat, high or low pH, or biochemical degradation by endogenous and exogenous RNAses. Intactness of RNA is crucial for a number of applications like polymerase-mediated linear amplification, Northern analysis, RNase protection assays and microarray analysis with standard methods.
Treatment of RNA with aldehyde fixatives such as formaldehyde (paraformaldehyde, formalin) causes chemical modification (addition of methylol groups to amino groups) in RNA, DNA and proteins and intra- and intermolecular crosslinking of RNA strands and crosslinking of RNA with protein through methylene bridges. Treatment of tissue with fixatives like formaldeyde or paraformaldehyde compromises the isolation of RNA from tissue using standard protocols like chaotropic agents or phenol-based methods. Efficient extraction can only be achieved when proteolytic enzymes like proteinase K or other proteases are used to digest crosslinked proteins into small peptides. Nucleic acids become soluble, although small peptides may remain attached (cross-linked). Proteolytic digestion with protease does not usually destroy crosslinks and chemical modifications in RNA, and methylol groups which are bound to amino groups of nucleobases remain preserved. Some procedures have been described to partially revert these modifications, but a major challenge remains that demodification and recovery of RNA from archival material are highly variable, and down-stream applications are sensitive towards varying amounts of residual modifications.
Usually, partial fragmentation of RNA by the action of endogenous RNases is not an important issue because the material is normally processed for histological analyses, which are not affected by this process and therefore, no special precautions are taken to reduce or prevent RNA degradation. In many situations, the starting material is an archival sample, which has been prepared earlier in the context of routine diagnosis or in the context of clinical trials. RNA prepared from routinely processed tissue is in the range of several hundred nucleotides, and only a small fraction of RNA comprises less than 100 nucleotides. Degradation of RNA to this size does not greatly affect methods like qRT-PCR, and therefore, RNA from archival material might be a perfect substrate for gene expression measurement when carried out with gene-specific primers during reverse transcription, and when PCR is performed with primers coding for amplicons which are smaller than hundred base pairs.
The use of high recoveries of RNA is fundamental for performing various molecular biological assays and experiments, such as normal RT-PCR, qRT-PCR and microarray experiments. The intrinsic instability of RNA and the presence of endogenous RNases in tissues makes the isolation of intact RNA a difficult procedure, but partially degraded RNA can be isolated. Although the contamination of molecular biology laboratories with RNases is usually not the major source of low quality RNA in this context, there is an ongoing need to develop improved techniques, which make RNA isolation and detection assay methods more sensitive, more specific, faster, and less susceptible to partial degradation. Ideally, it would be advantageous for research facilities in many instances to use an automated RNA isolation protocol, in order to combine it with rapid RNA assay techniques or integrated nucleic acid diagnostic devices for efficient, automated RNA isolation and analysis.
All the current protocols are based on reagents and protocols to minimize RNA degradation by endogenous and exogenous RNases, but they do not usually use reagents to eliminate chemical cross-links and modifications in fixed RNA.
For example, Danenberg et al. (US 2006/0199197) present a protocol which provides an RNA suitable for reverse transcription and PCR. The protocol involves a guanidinium-containing buffer and heating to 70-90° C. Schlumpberger et al. (WO2007/068764) describe RNA isolation with a nucleophilic reagent and a heat treatment step, which should improve recovery of RNA and accessibility of the RNA for reverse transcription followed by PCR.
In view of the above, there is a need for methods and reagents that allow one to recover at high efficiency RNA (including partially degraded RNA) from tissue samples treated with formalin and embedded in paraffin followed by storage at ambient or near ambient temperature for extended periods of time.
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