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. 2007 Oct;13(10):1668-74.
doi: 10.1261/rna.642907. Epub 2007 Aug 13.

Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples

Yaguang Xi  1 , Go Nakajima, Elaine Gavin, Chris G Morris, Kenji Kudo, Kazuhiko Hayashi, Jingfang Ju
Affiliations

Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples

Yaguang Xi et al. RNA. 2007 Oct.

Abstract

microRNAs (miRNAs) are noncoding small RNAs that regulate gene expression at the translational level by mainly interacting with 3' UTRs of their target mRNAs. Archived formalin-fixed paraffin-embedded (FFPE) specimens represent excellent resources for biomarker discovery. Currently there is a lack of systematic analysis on the stability of miRNAs and optimized conditions for expression analysis using FFPE samples. In this study, the expression of miRNAs from FFPE samples was analyzed using high-throughput locked nucleic acid-based miRNA arrays. The effect of formalin fixation on the stability of miRNAs was also investigated using miRNA real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The stability of miRNAs of archived colorectal cancer FFPE specimens was characterized with samples dating back up to 10 yr. Our results showed that the expression profiles of miRNAs were in good correlation between 1 mug of fresh frozen and 1-5 mug of FFPE samples (correlation coefficient R (2) = 0.86-0.89). Different formalin fixation times did not change the stability of miRNAs based on real-time qRT-PCR analysis. There are no significant differences of representative miRNA expression among 40 colorectal cancer FFPE specimens. This study provides a foundation for miRNA investigation using FFPE samples in cancer and other types of diseases.

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Figures

FIGURE 1.
FIGURE 1.
Schematic illustrations of sample processing and miRNA expression analysis from mouse liver tissue.
FIGURE 2.
FIGURE 2.
Effect of formalin fixation on miRNA expression. (A) Representative RNA agarose gel image (lane 1, RNA ladder; lane 2, fresh frozen sample; lane 3, FFPE sample). (B) The expression of hsa-miR-16 and RUN6B was analyzed using real-time QRT-PCR analysis from formalin-fixed mouse liver samples.
FIGURE 3.
FIGURE 3.
Correlation analysis of the global miRNAs expression between fresh frozen and FFPE samples. (A–C) The comparisons of different input RNA amounts (1, 3, and 5 μg) of FFPE samples versus 1 μg fresh frozen sample.
FIGURE 4.
FIGURE 4.
(A–C) Correlation analysis of the global miRNAs expression between different amounts of total RNA input (1, 3, and 5 μg) from FFPE samples.
FIGURE 5.
FIGURE 5.
Correlation analysis of the messenger RNA expression between fresh frozen and FFPE samples. (A) Global mRNAs expression comparison. (B) Overlapping mRNAs expression comparison.
FIGURE 6.
FIGURE 6.
Quantitative real-time PCR expression analysis of 5S (A) and miR-181b (B) from archived clinical colon cancer specimens. The line represents the trend of median expression levels of miRNAs in FFPE samples from different years. P-value was calculated by Wilcoxon's signed-rank sum test.
FIGURE 7.
FIGURE 7.
Representative miRNA array image from a 10-yr-old FFPE colon cancer sample.
See this image and copyright information in PMC

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