Ambion’s new RecoverAll™ Total Nucleic Acid Isolation Kit (patent pending) is designed for quantitative recovery of RNA and/or DNA from FFPE samples. The nucleic acid from the FFPE samples includes the full complement of microRNA (miRNA). These miRNAs can be used for miRNA expression profiling when used in combination with the flashPAGE™ Fractionator System and mirVana™ miRNA Array Technology.
Isolate microRNA from FFPE Samples
Total RNA (1 µg) from representative tissue samples was also analyzed on a Northern blot hybridized with a probe specific for let-7 (Figure 1). The recovery and quality of the let-7 miRNA in the FFPE and frozen samples were very similar, indicating that the new FFPE RNA isolation procedure was effective in recovering intact miRNA.
Figure 1. let-7 miRNA from FFPE and Flash-frozen Mouse Tissue Samples. Total RNA was isolated from four different mouse tissues that had been frozen (F) or formalin-fixed and paraffin-embedded (Z). This Northern blot (1 µg RNA/sample) was incubated with an oligonucleotide probe specific for let-7 and verified the presence of intact let-7 miRNA.
Obtain Reproducible miRNA Array Results
Figure 2. Excellent Correlation of miRNA Expression Profiles from FFPE and Flash-frozen Samples. For each tissue type, the R/G Normalized (Mean) ratio of each set of 4 fixed samples and 4 frozen samples was averaged. The average R/G ratio of the FFPE sample (X axis) was plotted against the average R/G ratio of the frozen sample (Y axis) to determine the correlation between miRNA profiles generated from fixed tissue samples as compared to frozen tissue samples ( A. Kidney, B. Liver, C. Brain). D. Representative heat maps from fixed (FFPE, left) and frozen (right) samples from brain.
Detect Differentially Expressed miRNAs
Figure 3. Similar Set of miRNAs Were Found to Be Differentially Expressed in Different Tissues in Fixed and Frozen Samples. The R/G Normalized (Mean) ratio of each set (n=4) of fixed and frozen samples was averaged for kidney and brain tissues. Panel A. The kidney to brain expression ratio for both fixed and frozen samples was determined, and the expression ratio of the fixed kidney vs. fixed brain (X axis) was plotted against the expression ratio of the frozen kidney vs. frozen brain (Y axis). Panel B. Representative examples of miRNA differentially express by at least 2-fold in kidney vs. brain are shown.
Examine Global miRNA Expression in FFPE Tissues
Emily Zeringer, Patricia Powers, Jaclyn Shingara, Kerri Keiger, Tim Barta, Rick Conrad, David Brown • Ambion, Inc.