Limits...
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.

Show MeSH

Related in: MedlinePlus

Comparison of miR abundance in paired fresh-frozen and FFPE tissues, using different RNA extraction methods. Plots show the comparison of Ct values for three normal and three mantle cell lymphoma samples by the three different extraction methods: A) FFPE (RecoverAll) vs. mirVana; B) FFPE (RecoverAll) vs. TRIzol; C) TRIzol vs. mirVana. Red lines delineate the abundance strata for each extraction method, with numbers indicating the average number of miRs per sample in each zone. For example, in the FFPE vs. mirVana plot (Figure 5A), 44 miRs are high abundance by mirVana and medium abundance by FFPE (RecoverAll), but only 4 miRs are high abundance by FFPE (RecoverAll) and medium abundance by mirVana.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2902407&req=5

Figure 5: Comparison of miR abundance in paired fresh-frozen and FFPE tissues, using different RNA extraction methods. Plots show the comparison of Ct values for three normal and three mantle cell lymphoma samples by the three different extraction methods: A) FFPE (RecoverAll) vs. mirVana; B) FFPE (RecoverAll) vs. TRIzol; C) TRIzol vs. mirVana. Red lines delineate the abundance strata for each extraction method, with numbers indicating the average number of miRs per sample in each zone. For example, in the FFPE vs. mirVana plot (Figure 5A), 44 miRs are high abundance by mirVana and medium abundance by FFPE (RecoverAll), but only 4 miRs are high abundance by FFPE (RecoverAll) and medium abundance by mirVana.

Mentions: Finally, the number of detectable miRs in each abundance stratum was determined for FFPE compared to fresh-frozen samples, using the results from all 3 normal benign lymph nodes and all 3 mantle cell lymphoma samples (Table 1 and Figure 5A-C). From this analysis, we detected a similar number of medium abundance miRs regardless of tissue source and extraction method used. Similar numbers of miRs are seen in all abundance strata among fresh-frozen samples regardless of extraction method used, whereas FFPE samples tend to have an increase in the number of low abundance miRs and a decreased number of high abundance miRs. Chi-square analysis demonstrates that the shift in proportion of miRs in the three strata is significantly different between fresh-frozen and FFPE tissues [TRIzol and FFPE (χ2 = 17.5, df = 2, p = 0.0002); mirVana and FFPE (χ2 = 13.5, df = 2, p = 0.001)], but not between TRIzol and mirVana (χ2 = 0.32, df = 2, p = 0.85). This difference may be due to reduced RNA yields in FFPE samples, which is known to occur and has been reported in the literature [7-10].


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)

Comparison of miR abundance in paired fresh-frozen and FFPE tissues, using different RNA extraction methods. Plots show the comparison of Ct values for three normal and three mantle cell lymphoma samples by the three different extraction methods: A) FFPE (RecoverAll) vs. mirVana; B) FFPE (RecoverAll) vs. TRIzol; C) TRIzol vs. mirVana. Red lines delineate the abundance strata for each extraction method, with numbers indicating the average number of miRs per sample in each zone. For example, in the FFPE vs. mirVana plot (Figure 5A), 44 miRs are high abundance by mirVana and medium abundance by FFPE (RecoverAll), but only 4 miRs are high abundance by FFPE (RecoverAll) and medium abundance by mirVana.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Comparison of miR abundance in paired fresh-frozen and FFPE tissues, using different RNA extraction methods. Plots show the comparison of Ct values for three normal and three mantle cell lymphoma samples by the three different extraction methods: A) FFPE (RecoverAll) vs. mirVana; B) FFPE (RecoverAll) vs. TRIzol; C) TRIzol vs. mirVana. Red lines delineate the abundance strata for each extraction method, with numbers indicating the average number of miRs per sample in each zone. For example, in the FFPE vs. mirVana plot (Figure 5A), 44 miRs are high abundance by mirVana and medium abundance by FFPE (RecoverAll), but only 4 miRs are high abundance by FFPE (RecoverAll) and medium abundance by mirVana.
Mentions: Finally, the number of detectable miRs in each abundance stratum was determined for FFPE compared to fresh-frozen samples, using the results from all 3 normal benign lymph nodes and all 3 mantle cell lymphoma samples (Table 1 and Figure 5A-C). From this analysis, we detected a similar number of medium abundance miRs regardless of tissue source and extraction method used. Similar numbers of miRs are seen in all abundance strata among fresh-frozen samples regardless of extraction method used, whereas FFPE samples tend to have an increase in the number of low abundance miRs and a decreased number of high abundance miRs. Chi-square analysis demonstrates that the shift in proportion of miRs in the three strata is significantly different between fresh-frozen and FFPE tissues [TRIzol and FFPE (χ2 = 17.5, df = 2, p = 0.0002); mirVana and FFPE (χ2 = 13.5, df = 2, p = 0.001)], but not between TRIzol and mirVana (χ2 = 0.32, df = 2, p = 0.85). This difference may be due to reduced RNA yields in FFPE samples, which is known to occur and has been reported in the literature [7-10].

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