- Optimized for isolation of total nucleic acids, including microRNAs, from FFPE tissue
- No overnight Proteinase K digestion required—deparaffinize in the morning and perform qRT-PCR in the afternoon
- Obtain typical yields of >50% that of unfixed tissue from the same sample source
- Recovered nucleic acids are suitable for qRT-PCR, qPCR, mutation screening, and microarray analyses
The RecoverAll Total Nucleic Acid Isolation Kit is designed to extract total nucleic acid from formaldehyde or paraformaldehyde-fixed, paraffin-embedded (FFPE) tissues (see schematic at right). Up to four 20 µm sections, or up to 35 mg of unsectioned core samples, can be processed per reaction.
The ability to isolate nucleic acid from archived tissue samples that is suitable for molecular analysis enables retrospective studies of diseased tissue at both the genomic and gene expression level. While standard preservation techniques that employ formaldehyde are ideal for maintaining tissue structure and preventing putrifecation, this type of preservation makes it difficult to perform molecular analyses on samples. Nucleic acids are trapped and modified through protein-protein and protein-nucleic acid crosslinks. In addition, RNA (and to some extent DNA) is often fragmented and chemically modified to such a degree that it is incompatible with many molecular analysis techniques.
The degree of RNA fragmentation that has already occurred in FFPE tissues cannot be reversed. However, the protease digestion conditions of the RecoverAll Kit are designed to release a maximal amount (see Figure 3) of RNA fragments of all sizes, including microRNA, in a relatively short amount of time.
The RecoverAll Total Nucleic Acid Isolation Kit procedure requires about 45 minutes of hands-on time and can easily be completed in less than 1 day when isolating RNA. FFPE samples are deparaffinized using a series of xylene and ethanol washes. Next, they are subjected to a rigorous protease digestion with an incubation time tailored for recovery of either RNA or DNA. The nucleic acids are purified using a rapid glass-filter methodology that includes an on-filter nuclease treatment and are eluted into either water or the low salt buffer provided.
The recovered nucleic acids are suitable for downstream applications such as microarray analyses, qRT-PCR (see Figure 4), and mutation screening. However, as is the case with all FFPE tissue, sample fixation and storage typically cause nucleic acid fragmentation and modification. Therefore, downstream applications, such as microarray analysis, which require more pristine RNA than does qRT-PCR, may require modification for best results.
Although DNA tends not to fragment as easily as RNA, it appears to be more reactive to the formaldehyde and requires a longer (2 day) protease digestion time to release substantial amounts of DNA. The recovered DNA can typically be used for PCR and other downstream applications.
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Product Comparison Article: Optimization of RNA extraction from FFPE tissues for expression profiling in the DASL assay.
BioTechniques 44:417-423.
[View Article] |
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