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

Goswami RS, Waldron L, Machado J, Cervigne NK, Xu W, Reis PP, Bailey DJ, Jurisica I, Crump MR, Kamel-Reid S - BMC Biotechnol. (2010)

Bottom Line: By dividing the profiled miR into abundance strata of high (Ct<30), medium (30 < or = Ct < or = 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.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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Applied Molecular Oncology, Ontario Cancer Institute, University Health Network, Toronto, ON, Canada.

ABSTRACT

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 < or = Ct < or = 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.

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Related in: MedlinePlus

Correlation heatmaps and PCA mapping for paired fresh-frozen and FFPE tissues. Correlation heat-maps demonstrating hierarchical clustering among 3 normal lymph nodes (n1-n3) and 3 mantle cell lymphomas (t1-t3) extracted using different techniques using: A) Low abundance miRs B) Medium abundance miRs C) High abundance miRs. Panel D) shows principal component analysis (PCA) confirming the similarity of miR expression between tumours (red), and normal samples (blue), irrespective of tissue origin (FFPE vs. fresh-frozen).
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Figure 4: Correlation heatmaps and PCA mapping for paired fresh-frozen and FFPE tissues. Correlation heat-maps demonstrating hierarchical clustering among 3 normal lymph nodes (n1-n3) and 3 mantle cell lymphomas (t1-t3) extracted using different techniques using: A) Low abundance miRs B) Medium abundance miRs C) High abundance miRs. Panel D) shows principal component analysis (PCA) confirming the similarity of miR expression between tumours (red), and normal samples (blue), irrespective of tissue origin (FFPE vs. fresh-frozen).

Mentions: Unsupervised hierarchical clustering analysis was performed (hclust R function, one minus Pearson correlation distance measure) for tumours and normal samples, taking into account the extraction method used. Figures 4A-C are heatmaps showing the results for the low, medium and high abundance miRs. Principal component analysis (PCA) was also applied, and confirmed that all tumours group together and separate from normal samples, independent of the tissue specimen and method used for RNA extraction (Figure 4D).


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

Goswami RS, Waldron L, Machado J, Cervigne NK, Xu W, Reis PP, Bailey DJ, Jurisica I, Crump MR, Kamel-Reid S - BMC Biotechnol. (2010)

Correlation heatmaps and PCA mapping for paired fresh-frozen and FFPE tissues. Correlation heat-maps demonstrating hierarchical clustering among 3 normal lymph nodes (n1-n3) and 3 mantle cell lymphomas (t1-t3) extracted using different techniques using: A) Low abundance miRs B) Medium abundance miRs C) High abundance miRs. Panel D) shows principal component analysis (PCA) confirming the similarity of miR expression between tumours (red), and normal samples (blue), irrespective of tissue origin (FFPE vs. fresh-frozen).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2902407&req=5

Figure 4: Correlation heatmaps and PCA mapping for paired fresh-frozen and FFPE tissues. Correlation heat-maps demonstrating hierarchical clustering among 3 normal lymph nodes (n1-n3) and 3 mantle cell lymphomas (t1-t3) extracted using different techniques using: A) Low abundance miRs B) Medium abundance miRs C) High abundance miRs. Panel D) shows principal component analysis (PCA) confirming the similarity of miR expression between tumours (red), and normal samples (blue), irrespective of tissue origin (FFPE vs. fresh-frozen).
Mentions: Unsupervised hierarchical clustering analysis was performed (hclust R function, one minus Pearson correlation distance measure) for tumours and normal samples, taking into account the extraction method used. Figures 4A-C are heatmaps showing the results for the low, medium and high abundance miRs. Principal component analysis (PCA) was also applied, and confirmed that all tumours group together and separate from normal samples, independent of the tissue specimen and method used for RNA extraction (Figure 4D).

Bottom Line: By dividing the profiled miR into abundance strata of high (Ct<30), medium (30 < or = Ct < or = 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.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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Applied Molecular Oncology, Ontario Cancer Institute, University Health Network, Toronto, ON, Canada.

ABSTRACT

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 < or = Ct < or = 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.

Show MeSH
Related in: MedlinePlus