Optimization and analysis of a quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples

被引：34

作者：

Goswami, Rashmi S. ^{[1
,2
]}

Waldron, Levi ^{[3
,4
]}

Machado, Jerry ^{[1
,2
]}

Cervigne, Nilva K. ^{[1
]}

Xu, Wei ^{[5
]}

Reis, Patricia P. ^{[1
]}

Bailey, Denis J. ^{[2
,6
]}

Jurisica, Igor ^{[3
,7
,8
]}

Crump, Michael R. ^{[9
]}

Kamel-Reid, Suzanne ^{[1
,2
,6
,8
]}

机构：

[1] Univ Hlth Network, Div Appl Mol Oncol, Ontario Canc Inst, Toronto, ON, Canada

[2] Univ Toronto, Dept Lab Med & Pathobiol, Toronto, ON, Canada

[3] Univ Hlth Network, Div Signaling Biol, Ontario Canc Inst, Toronto, ON, Canada

[4] Univ Hlth Network, Campbell Family Inst Canc Res, Toronto, ON, Canada

[5] Univ Hlth Network, Dept Biostat, Princess Margaret Hosp, Toronto, ON, Canada

[6] Univ Hlth Network, Dept Pathol, Toronto Gen Hosp, Toronto, ON, Canada

[7] Univ Toronto, Dept Comp Sci, Toronto, ON, Canada

[8] Univ Toronto, Dept Med Biophys, Toronto, ON, Canada

[9] Univ Hlth Network, Div Med Oncol, Princess Margaret Hosp, Toronto, ON, Canada

来源：

BMC BIOTECHNOLOGY | 2010年 / 10卷

基金：

加拿大创新基金会;

关键词：

MANTLE CELL LYMPHOMA; RNA; SIGNATURE; BIOLOGY; TISSUES; FROZEN;

D O I：

10.1186/1472-6750-10-47

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 [微生物学]; 090105 [作物生产系统与生态工程];

摘要：

Background: MicroRNAs (miRs) are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE) tissue. Results: Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access) platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p < 0.00001) and outlines the optimal performance conditions of this platform using clinical FFPE samples. We also outline a method of data analysis looking at differences in miR abundance between FFPE and fresh-frozen samples. By dividing the profiled miR into abundance strata of high (Ct<30), medium (30 <= Ct <= 35), and low (Ct>35), we show that reproducibility between technical replicates, equivalent dilutions, and FFPE vs. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p < 0.001), when examining all miRs, regardless of RNA extraction method used. Examining correlation coefficients between FFPE and fresh-frozen samples in terms of miR abundance reveals correlation coefficients of up to 0.32 (low abundance), 0.70 (medium abundance) and up to 0.97 (high abundance). Conclusion: Our study thus demonstrates the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies.

引用

页数：12

共 32 条

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