The RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE 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 20μm sections, or up to 35mg of unsectioned, core samples each.
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 use 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 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 one 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 (two-day) protease digestion time to release substantial amounts of DNA.
- 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 next generation sequencing, real-time RT-PCR, PCR, mutation screening, and microarray analyses
Not high-throughput compatible (Manual)
DNA and RNA Purification and Analysis, RNA Extraction, Specialty Nucleic Acid Purification (e.g. Total Nucleic Acids), Total RNA Isolation, Total RNA from FFPE Samples
|Genomic DNA, Total RNA, micro RNA|
|16mL Digestion Buffer; 60mL Wash 1 Concentrate; 60mL Wash 2/3 Concentrate; 80 Collection Tubes; 40 Filter Cartridges; 19.2mL Isolation Additive; 5mL Elution Solution; 160μL Protease; 240μL 10X DNase Buffer; 160μL DNase; 400μL RNase A|
|FFPE & Fixed Samples|
|Genomic DNA, Total Nucleic Acids, Total RNA, miRNA|
|Next-Generation Sequencing, Northern Blotting, PCR, RT-PCR (Endpoint), Real-Time PCR, Southern Blotting, cDNA Library Construction, microRNA Analysis|
|Spin Column (Glass Fiber Filter)|
|Up to 35mg of unsectioned core samples, Up to four 20μm sections|
|Digestion Buffer (room temperature); Wash 1 Concentrate (room temperature); Wash 2/3 Concentrate (room temperature); Collection Tubes (room temperature); Filter Cartridges (room temperature); Isolation Additive (room temperature); Elution Solution (−20°C, 4°C, or room temperature); Protease (-20°C); 10X DNase Buffer (−20°C); DNase (−20°C); RNase A (−20°C). This kit is shipped in two parts: one at room temperature and one at −20°C.|
For Research Use Only. Not for use in diagnostic procedures.