RNeasy FFPE Kit
For purification of total RNA from formalin-fixed, paraffin-embedded tissue sections
The RNeasy FFPE Kit is specially designed for purifying total RNA from formalin-fixed, paraffin-embedded tissue sections. Special lysis and incubation conditions reverse formaldehye modification of RNA. In addition, the lysis buffer efficiently releases RNA from tissue sections while avoiding further RNA degradation. The kit also uses DNase and DNase Booster Buffer for optimized removal of genomic DNA contamination. RNeasy MinElute spin columns enable purification of total RNA with elution volumes of as low as 10 μl. Purification can be fully automated on the QIAcube Connect.
With the RNeasy FFPE Kit, yields of RNA purified from fixed and embedded samples are greater than those achieved using other methods (see figure “Greater yields of RNA”). Recovered RNA is intact down to 70 nucleotides in length and virtually free of genomic DNA contamination (see figure “Recovery of all usable RNA”). Usable RNA fragments purified with the kit from FFPE samples are highly suited for use in sensitive downstream applications, including real-time RT-PCR (see figures “Reliable results in real-time RT-PCR analysis” and “Successful real-time RT-PCR analysis”).
Fixing tissues with formalin leads to RNA–RNA and RNA–protein crosslinking which impairs RNA performance in enzymatic assays. Fixation and embedding conditions can also result in heavily fragmented nucleic acids in FFPE samples. The RNeasy FFPE Kit uses special lysis and incubation conditions to reverse formaldehyde modification of RNA. The lysis buffer efficiently releases RNA from FFPE tissue samples, preventing further RNA degradation. These optimized conditions allow purification of all usable RNA, leading to greater yields from FFPE samples with the RNeasy FFPE Kit than with other methods.
The entire RNeasy FFPE procedure can be completed in as little as 85 minutes (see flowchart "RNeasy FFPE procedure"). Sample lysis with proteinase K digestion requires only 15 minutes. After lysis, samples are incubated at 80ºC for 15 minutes. DNase treatment then effectively removes genomic DNA, including small DNA fragments. Finally, concentrated RNA is purified using RNeasy MinElute spin columns, and eluted in a volume of 14–30 µl. Purification can be fully automated on the QIAcube.
The RNeasy FFPE Kit isolates all RNA molecules longer than 70 nucleotides from FFPE samples, providing usable RNA fragments for numerous downstream applications, including RT-PCR. However, RNA purified from FFPE samples is heavily fragmented and should not be used in downstream applications that require full-length RNA. Some applications may require modifications to allow the use of fragmented RNA (e.g., designing small amplicons for RT-PCR). For cDNA synthesis, either random or gene-specific primers should be used instead of oligo-dT primers.
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