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 the 40 purifications from up to four 20 μm sections, or up to 35 mg of unsectioned core samples each.
• 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 real-time RT-PCR, PCR, mutation screening, and microarray analyses
Archived tissue samples contain valuable information of disease states; however 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 查看更多putrefecation, but 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 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 and are eluted into either water or the low salt buffer provided.
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 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 formalin and requires a longer (2 day) protease digestion time to release substantial amounts of DNA.
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