The Ambion® RecoverAll™ Total Nucleic Acid Isolation Kit is for the extraction of total nucleic acid from formalin- or paraformalin-fixed, paraffin-embedded (FFPE) tissues. Sufficient reagents are included for 40 purifications from up to four 20 µm sections, or up to 35 mg of unsectioned core samples each.
Features of the RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE:
• 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
• Typical yields are >50% of those of unfixed tissue
• Recovered nucleic acids are suitable for real-time RT-PCR, PCR, mutation screening, and microarray analyses
Extraction of Nucleic Acids from Difficult Samples
Archived tissue samples contain valuable information of disease states, but it has traditionally been difficult to isolate nucleic acids from them of a quality suitable for molecular analysis. Standard preservation techniques employ formalin that maintains tissue structure and prevents putrefaction, but which also traps nucleic acids and modifies them through protein-protein and protein-nucleic acid crosslinks. RNA (and to some extent DNA) is often so fragmented and chemically modified that it is incompatible with many molecular analysis techniques. RNA fragmentation in FFPE tissues cannot be reversed; however, the protease digestion conditions of the RecoverAll™ Kit are designed to release a maximal amount (see Figure) of trapped RNA fragments of all sizes, including microRNA, in a relatively short amount of time.
The RecoverAll™ Total Nucleic Acid Isolation Kit Solution
The RecoverAll™ Total Nucleic Acid Isolation Kit procedure requires about 45 minutes of hands-on time and can be completed in typically less than 1 day for RNA. FFPE samples are deparaffinized using a series of xylene and ethanol washes. Next, they are subjected to an extensive protease digestion with an incubation time tailored for the recovery of either RNA or DNA. Nucleic acids are then purified using a rapid glass-filter method, and are eluted into either water or low-salt buffer.
Nucleic Acids for Almost Any Downstream Application
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 optimal results. Although DNA tends not to fragment as easily as RNA, it appears to be more reactive to the formalin and requires a longer (2 day) protease digestion time to release substantial amounts of DNA. For research use only. Not for use in diagnostic procedures.