This study describes the successful isolation and accurate measurement of miRNA expression from formalin- or paraformalin-fixed, paraffin-embedded (FFPE) samples. miRNAs are robustly and reproducibly amplified from FFPE samples using TaqMan® MicroRNA Assays. Comparison to matched, snap-frozen samples demonstrates high concordance between the data generated from the two sample sources, confirming that the FFPE fixation process does not adversely affect miRNA quantitation.

Unlocking the Potential of microRNAs in FFPE Tissues

It has recently been discovered that microRNAs (miRNAs), a class of small noncoding RNAs, are responsible for significant post-transcriptional regulation of gene expression. As publications describing miRNA function and targets accumulate, it is becoming clear that miRNAs are involved in regulating diverse biological processes, including cell development, differentiation, apoptosis, and proliferation [1]. There have been many inquiries into the possible role of miRNAs in cancer. Several recent publications document the differential expression of miRNAs between malignant and normal tissues, indicating that miRNAs play a role in tumor formation and that their expression patterns may prove useful for tumor classification and treatment [2–4].

FFPE tissues are valuable samples for the study of human cancer, since they are generally retrieved with extensively documented clinico-pathological histories. Isolating nucleic acids from archived samples has the potential to unlock a wealth of additional information, facilitating the study of human cancer at the molecular level.

While standard methods of preservation using formalin and paraformalin are ideal for maintaining tissue structure and preventing putrefaction, they pose challenges for the molecular analyses of these samples. Nucleic acids become trapped and modified through protein-nucleic acid and nucleic acid-nucleic acid cross-links. RNA isolated from FFPE samples is often fragmented to a random range of sizes and chemically modified to a degree that is incompatible with many molecular analysis techniques. To address this challenge, Applied Biosystems scientists have developed an optimized protocol for the extraction and quantitation of RNA from FFPE tissues [5].

Comparison of microRNA Profiles from FFPE and Snap-Frozen Human Tissues

Dr. Leendert H.J. Looijenga and colleagues, in the Department of Pathology at the Erasmus MC-University Medical Center in The Netherlands, analyzed miRNA expression in archived human germ-cell tumors using high throughput real-time PCR [6]. Their results support recent observations that miRNAs are involved in regulating stem cell differentiation [7]. To confirm that results were not affected by the FFPE fixation process, the researchers performed a comparative study using matched testis and seminoma samples that were either preserved by FFPE fixation or snap-frozen immediately upon collection. Total RNA was extracted from FFPE samples using the Ambion® RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE, and from snap-frozen samples using the Ambion® mirVana™ miRNA Isolation Kit.

In this experiment, four different small RNAs (5S rRNA, U6 snRNA, hsa-miR-372 and hsa-miR-373) were assayed in the samples using TaqMan MicroRNA Assays which contain both a small RNA-specific stem-loop RT primer and a PCR primer/TaqMan probe mix (Figure 1). Reverse transcription was performed using identical amounts of input total RNA from matched snap-frozen and FFPE samples. A portion of the resulting cDNA amplified using real-time PCR produced highly similar CT values (Figure 1).

Figure 1. Highly Comparable Results of miRNA Quantitation in FFPE Tissues and Snap-Frozen Samples. Total RNA was extracted using the Ambion® mirVana™ miRNA Isolation Kit for the snap-frozen samples and the RecoverAll™ Kit for the FFPE tissues. Individual small RNAs were detected by real-time PCR using TaqMan® MicroRNA Assays. The mean CT values of triplicates are shown on the y-axis. [Data courtesy of Leendert Looijenga and Ad Gillis, Erasmus MC-University Medical Center Rotterdam, The Netherlands]

In a similar, but larger study, Dr. Orla Sheils and colleagues from the Department of Histopathology at the University of Dublin, Ireland, compared the expression profiles of 160 miRNAs in matched samples of snap-frozen and FFPE cells (cells isolated from FFPE tissues by laser capture microdissection) [8]. Total RNA was isolated from ~2 x 106 FFPE-preserved thyroid cancer cells and from ~1.7 x 105 snap-frozen cells using the RecoverAll Total Nucleic Acid Isolation Kit for FFPE and the mirVana miRNA Isolation Kit, respectively. As in the first study, Applied Biosystems TaqMan MicroRNA Assays were used for miRNA expression profiling by this group. The real-time PCRs were run in triplicate for each miRNA quantitated (Figure 2).

Figure 2. High Correlation of Mean CT Values of 154 miRNA Assays from Paired FFPE and Snap-Frozen Cells. RNA was extracted from snap-frozen cells using the Ambion® mirVana™ miRNA Isolation Kit and from FFPE cells using RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE. Identical amounts of total RNA were used in TaqMan® MicroRNA Assays. Real-time PCRs were run in triplicate. The mean CT values of the FFPE samples are shown on the y-axis and the mean CT values of the snap-frozen cells are shown on the x-axis. [Data courtesy of Orla Sheils, University of Dublin, Ireland]

The results presented in both studies demonstrate a high degree of correlation of miRNA detection irrespective of the method for preserving the tissue, confirming that miRNA expression levels are well preserved in FFPE samples. Furthermore, these results demonstrate the effectiveness of Applied Biosystems research tools for both miRNA recovery and analysis in FFPE samples.

Reproducibility of miRNA Detection in FFPE Samples

Prof Looijenga performed a follow-up study to confirm the reproducibility of the detected miRNA expression levels in FFPE tissues across a small population. For this purpose, the expression levels of 157 miRNAs in five biological replicates of yolk sac tumor FFPE samples, each resected from a different individual, were compared. Total RNA was extracted from the FFPE samples with the RecoverAll Kit and identical amounts were analyzed using TaqMan MicroRNA Assays. The results show that miRNAs are reproducibly quantitated across biological replicates from FFPE tissues (Figure 3).

Figure 3. Expression Levels of 23 Representative miRNAs in 5 Biological Replicates of Yolk Sac Tumor FFPE Samples (YS-1 to YS-5). RNA was isolated from 8–20 year old FFPE tumor samples with the Ambion® RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE Tissues according to the protocol provided with the kit. A total of 157 miRNAs were analyzed per sample. Mean CT values from triplicate PCRs are shown for some representative miRNAs. [Data courtesy of Leendert Looijenga and Ad Gillis, Erasmus MC-University Medical Center Rotterdam, The Netherlands]

In conclusion, these studies clearly demonstrate a high correlation of miRNA expression patterns between FFPE and matched snap-frozen cells (Figures 1 and 2). The analysis of biological replicates illustrates the reproducibility of miRNA detection from FFPE samples (Figure 3). A general workflow to extract and amplify miRNA from FFPE tissues is shown in Figure 4.

Figure 4. Applied Biosystems Workflow for MicroRNA Extraction and Quantitation from FFPE Tissues.

Benefits of miRNA Analysis from FFPE Tissues

The detection of messenger RNAs from archival material has been challenging due to the labile nature of RNA, and the deleterious effects of enzymatic fragmentation and/or RNA modification induced by formalin fixation. As a potential solution, it has been suggested that small PCR amplicons (shorter than 130 nucleotides) could have utility as robust markers in gene expression studies using FFPE tissues [5].

In contrast, mature miRNAs have the inherent advantage of small size (20–22 nucleotides), and it is postulated that they may present a difficult target for enzymatic or chemical cleavage. In addition, miRNAs are thought to be further protected from the effects of formalin fixation by their tight association with proteins of the RNA-induced silencing complex (RISC). This hypothesis was recently supported by data indicating that miRNAs are tightly associated with RISC in vivo [9]. Tang and co-workers used real-time PCR to determine the proportion of miRNAs that were associated stably with RISCs in vivo under physiological conditions. The authors concluded that only 1–3% of mature miRNAs were free in cells.

Irrespective of the mechanisms involved, these data suggest that, compared to mRNA, miRNAs are not as susceptible to the deleterious effects of fixation associated with FFPE processing and, as a consequence, represent a set of new and promising biomarkers with potential to unlock the wealth of information in FFPE sample collections.

Scientific Contributors
Leendert H.J. Looijenga • Erasmus MC-University Medical Center, The Netherlands
Orla Sheils • University of Dublin, Ireland
Astrid Ferlinz, Jihad Skaf, Iain Russell, Simone Guenther • Applied Biosystems