Applied Biosystems has improved the protocol for the Ambion® RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE Tissues, resulting in significantly reduced processing time. No reagents or formulations have changed—only pipetting volumes, incubation times, and temperatures. The end result is a shortened protocol with equivalent nucleic acid yield and performance to that of the original protocol.

FFPE Tissues Abundant but Challenging

Many standard preservation techniques for biological tissue sample storage employ formalin or paraformalin to maintain tissue structure and prevent putrefaction. However, this type of preservation makes it difficult to perform molecular analysis on the samples since the nucleic acids are both trapped and modified by significant protein-protein and protein-nucleic acid crosslinks. Researchers at Applied Biosystems have learned that the fixation process itself does not necessarily lead to nucleic acid fragmentation [1]. Instead, the embedding process, which requires high temperatures and vacuum for the paraffin to penetrate the tissue, quickens chemical reactions which subsequently modify RNA and DNA. These modifications lead to fragmentation over time.

A Faster Isolation Workflow

The Ambion RecoverAll Total Nucleic Acid Isolation Kit for FFPE Tissues efficiently isolates all nucleic acids, including miRNA, from formalin- or paraformalin-fixed, paraffin-embedded (FFPE) tissues. Up to four 20 µm sections, or up to 35 mg of unsectioned core samples, can be processed per reaction. Recent improvements to the kit protocol reduce hands-on time and simplify the workflow, so that the isolation of nucleic acids from FFPE tissue is easier, and 67% faster. Although the degree of RNA and DNA fragmentation that has already occurred in FFPE tissues cannot be reversed, nucleic acids extracted from FFPE tissues are still functional and can be analyzed by PCR. Modified protease digestion conditions used in the improved RecoverAll Kit protocol are designed to accelerate release of maximal amounts of nucleic acid in only 30 minutes for RNA, or overnight for DNA (Figure 1).

Figure 1. Faster RecoverAll™ Total Nucleic Acid Isolation Kit Workflow.

Faster Recovery Without Sacrificing Results

The improved RecoverAll workflow continues to deliver nucleic acids with high functionality in downstream applications. A study performed on breast, lung, and colon tissue showed that, although nucleic acid yield, based on absorbance readings at 260 nm, varied between blocks of FFPE tissues due to age, core size and tissue type, overall yield data with the new RecoverAll Kit was equivalent to that from the original kit (data not shown). The coefficient of variation for overall yield data from the original RecoverAll Kit protocol versus the improved protocol remains unchanged. Furthermore, the RNA isolated using the new protocol performs better in real-time PCR analysis (Figure 2). We propose that the shorter incubation time at higher temperatures results in purification of cleaner RNA that performs better in downstream reactions.

Figure 2. New, Faster RecoverAll™ Isolation Kit Protocol Gives RNA with Better Real-time PCR Performance than Original Protocol. Real-time RT-PCR was carried out for 4 targets (GAPDH, GUSB, miR-16 and miR-155) with RNA isolated from breast, lung and colon FFPE tissue using the new RecoverAll Isolation Kit protocol or the original protocol. In all cases, RNA isolated with the new protocol shows equivalent or lower CT values (better performance) than RNA isolated with the original protocol.

RecoverAll Kit Outperforms the Competition

The Ambion RecoverAll Total Nucleic Acid Isolation Kit for FFPE Tissues was compared to competitor FFPE kits. RNA was extracted from both breast and lung FFPE blocks using the Ambion RecoverAll Kit and kits from competitors Q, R, and S. RNA recovery from both sample types using the RecoverAll Kit, as measured by absorbance at 260 nm, was set at 100%. Recovery by the competitors’ kits ranged from 33−83% as compared to the RecoverAll Kit. Real-time PCR results corroborated this data, with equivalent or higher amounts of miRNA detected in the samples extracted using the RecoverAll Kit compared to the 3 competitors.

In addition, the Ambion RecoverAll Kit takes exactly 17 steps to complete the isolation of RNA from FFPE tissue samples with an overall RNA recovery time of 1–1.5 hr.

DNA Isolation with the RecoverAll Kit

The new Ambion RecoverAll Kit protocol provides instructions for both RNA and DNA recovery from FFPE treated tissue. Competitor Q offers separate kits to recover either RNA or DNA, while competitors R and S do not offer kits for DNA recovery.

Scientific Contributors

Emily Zeringer, Tim Barta, Rick Conrad • Applied Biosystems