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Impact of cellular miRNAs on circulating miRNA biomarker signatures.

Duttagupta R, Jiang R, Gollub J, Getts RC, Jones KW - PLoS ONE (2011)

Bottom Line: Correlations between tumor-associated genomic/epigenetic/transcriptional changes and alterations in CNA levels are strong predictors of the utility of this biomarker class as promising clinical indicators.We find that measurement of circulating miRNA levels are frequently confounded by varying levels of cellular miRNAs of different hematopoietic origins.Cellular miRNA signatures in cohorts of normal individuals are catalogued and the abundance and gender specific expression of bona fide circulating markers explored after calibrating the signal for this interfering class.

View Article: PubMed Central - PubMed

Affiliation: Applied Research, Affymetrix Inc, Santa Clara, California, United States of America.

ABSTRACT
Effective diagnosis and surveillance of complex multi-factorial disorders such as cancer can be improved by screening of easily accessible biomarkers. Highly stable cell free Circulating Nucleic Acids (CNA) present as both RNA and DNA species have been discovered in the blood and plasma of humans. Correlations between tumor-associated genomic/epigenetic/transcriptional changes and alterations in CNA levels are strong predictors of the utility of this biomarker class as promising clinical indicators. Towards this goal microRNAs (miRNAs) representing a class of naturally occurring small non-coding RNAs of 19-25 nt in length have emerged as an important set of markers that can associate their specific expression profiles with cancer development. In this study we investigate some of the pre-analytic considerations for isolating plasma fractions for the study of miRNA biomarkers. We find that measurement of circulating miRNA levels are frequently confounded by varying levels of cellular miRNAs of different hematopoietic origins. In order to assess the relative proportions of this cell-derived class, we have fractionated whole blood into plasma and its ensuing sub-fractions. Cellular miRNA signatures in cohorts of normal individuals are catalogued and the abundance and gender specific expression of bona fide circulating markers explored after calibrating the signal for this interfering class. A map of differentially expressed profiles is presented and the intrinsic variability of circulating miRNA species investigated in subsets of healthy males and females.

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Correlation of expression levels of circulating miRNAs across different biological replicates in the CS and S1 fractions.(A) Spearman's Rank Correlation coefficients for CS and S1 fractions, across all replicates restricting to the highest expressing 20, 50, 200 or 534 (all) human miRNAs that are common to the 2 fractions. Each point on the graph represents the rank correlation values across all pair-wise combination of replicates for the category under study. (B) Mean correlation values for the CS and S1 fraction in each intensity strata. (C) Analysis of Coefficient of Variance of the CS and S1 fractions for the 534 miRNAs under study.
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pone-0020769-g005: Correlation of expression levels of circulating miRNAs across different biological replicates in the CS and S1 fractions.(A) Spearman's Rank Correlation coefficients for CS and S1 fractions, across all replicates restricting to the highest expressing 20, 50, 200 or 534 (all) human miRNAs that are common to the 2 fractions. Each point on the graph represents the rank correlation values across all pair-wise combination of replicates for the category under study. (B) Mean correlation values for the CS and S1 fraction in each intensity strata. (C) Analysis of Coefficient of Variance of the CS and S1 fractions for the 534 miRNAs under study.

Mentions: In principal the process of sub-fractionation, while removing contaminants may potentially impact the stability of expression of the truly circulating miRNA species. In order to assess if the population of miRNAs detected in the CS and S1 fractions display consistent patterns of expression, we analyzed both the rank order concordance of miRNAs in the CS and S1 fractions across all biological replicates and the distribution of the Coefficient of Variance (CV) between these two fractions for all miRNAs within the replicates studied. We hypothesized that conserved and stably expressed miRNAs would have non-stochastic expression levels and hence demonstrate not only high rank order correlations but also low CV estimates. In order to test this, miRNAs were selected after filtering out contaminant features and a common set of targets isolated based on average intensities from the CS and S1 fractions across multiple biological replicates. Categories of miRNAs were stratified into intensity bins containing the highest expressing 20, 50, 200 or 534 miRNAs and Spearman's Rank Correlation values computed for each category per individual fraction across all samples (Fig. 5A). Our results show modest improvement in the mean correlation values for the CS fractions across the different intensity strata (Fig. 5B). In contrast we observe a statistically significant 2.5 fold increase in correlation in the S1 fraction as the analysis is restricted from the entire 534 features to the highest expressing top 20 miRNA class (P-value of 9.624552e-18 from two-sided Student's t-test). Additionally the comparison of variance between the different fractions across all replicates also reveal a statistically significant higher variance for the CS fraction compared to the S1 isolate for the entire spectrum of 534 miRNAs (p-values of 6.138508e-13 from Student's t-test) (Fig. 5C). Taken together these results indicate that the population of miRNAs in the S1 fraction is homogenously expressed across replicates with significantly lower variability than species measured in the CS fraction. Additionally the process of fractionation of CS into S1 tends to preserve the integrity of the higher expressed miRNAs with no additional negative impact on the lower abundant species. The S1 fraction hence represents a comprehensive plasma category enriched in circulating markers that can be reproducibly detected in replicate samples for biological analysis.


Impact of cellular miRNAs on circulating miRNA biomarker signatures.

Duttagupta R, Jiang R, Gollub J, Getts RC, Jones KW - PLoS ONE (2011)

Correlation of expression levels of circulating miRNAs across different biological replicates in the CS and S1 fractions.(A) Spearman's Rank Correlation coefficients for CS and S1 fractions, across all replicates restricting to the highest expressing 20, 50, 200 or 534 (all) human miRNAs that are common to the 2 fractions. Each point on the graph represents the rank correlation values across all pair-wise combination of replicates for the category under study. (B) Mean correlation values for the CS and S1 fraction in each intensity strata. (C) Analysis of Coefficient of Variance of the CS and S1 fractions for the 534 miRNAs under study.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3117799&req=5

pone-0020769-g005: Correlation of expression levels of circulating miRNAs across different biological replicates in the CS and S1 fractions.(A) Spearman's Rank Correlation coefficients for CS and S1 fractions, across all replicates restricting to the highest expressing 20, 50, 200 or 534 (all) human miRNAs that are common to the 2 fractions. Each point on the graph represents the rank correlation values across all pair-wise combination of replicates for the category under study. (B) Mean correlation values for the CS and S1 fraction in each intensity strata. (C) Analysis of Coefficient of Variance of the CS and S1 fractions for the 534 miRNAs under study.
Mentions: In principal the process of sub-fractionation, while removing contaminants may potentially impact the stability of expression of the truly circulating miRNA species. In order to assess if the population of miRNAs detected in the CS and S1 fractions display consistent patterns of expression, we analyzed both the rank order concordance of miRNAs in the CS and S1 fractions across all biological replicates and the distribution of the Coefficient of Variance (CV) between these two fractions for all miRNAs within the replicates studied. We hypothesized that conserved and stably expressed miRNAs would have non-stochastic expression levels and hence demonstrate not only high rank order correlations but also low CV estimates. In order to test this, miRNAs were selected after filtering out contaminant features and a common set of targets isolated based on average intensities from the CS and S1 fractions across multiple biological replicates. Categories of miRNAs were stratified into intensity bins containing the highest expressing 20, 50, 200 or 534 miRNAs and Spearman's Rank Correlation values computed for each category per individual fraction across all samples (Fig. 5A). Our results show modest improvement in the mean correlation values for the CS fractions across the different intensity strata (Fig. 5B). In contrast we observe a statistically significant 2.5 fold increase in correlation in the S1 fraction as the analysis is restricted from the entire 534 features to the highest expressing top 20 miRNA class (P-value of 9.624552e-18 from two-sided Student's t-test). Additionally the comparison of variance between the different fractions across all replicates also reveal a statistically significant higher variance for the CS fraction compared to the S1 isolate for the entire spectrum of 534 miRNAs (p-values of 6.138508e-13 from Student's t-test) (Fig. 5C). Taken together these results indicate that the population of miRNAs in the S1 fraction is homogenously expressed across replicates with significantly lower variability than species measured in the CS fraction. Additionally the process of fractionation of CS into S1 tends to preserve the integrity of the higher expressed miRNAs with no additional negative impact on the lower abundant species. The S1 fraction hence represents a comprehensive plasma category enriched in circulating markers that can be reproducibly detected in replicate samples for biological analysis.

Bottom Line: Correlations between tumor-associated genomic/epigenetic/transcriptional changes and alterations in CNA levels are strong predictors of the utility of this biomarker class as promising clinical indicators.We find that measurement of circulating miRNA levels are frequently confounded by varying levels of cellular miRNAs of different hematopoietic origins.Cellular miRNA signatures in cohorts of normal individuals are catalogued and the abundance and gender specific expression of bona fide circulating markers explored after calibrating the signal for this interfering class.

View Article: PubMed Central - PubMed

Affiliation: Applied Research, Affymetrix Inc, Santa Clara, California, United States of America.

ABSTRACT
Effective diagnosis and surveillance of complex multi-factorial disorders such as cancer can be improved by screening of easily accessible biomarkers. Highly stable cell free Circulating Nucleic Acids (CNA) present as both RNA and DNA species have been discovered in the blood and plasma of humans. Correlations between tumor-associated genomic/epigenetic/transcriptional changes and alterations in CNA levels are strong predictors of the utility of this biomarker class as promising clinical indicators. Towards this goal microRNAs (miRNAs) representing a class of naturally occurring small non-coding RNAs of 19-25 nt in length have emerged as an important set of markers that can associate their specific expression profiles with cancer development. In this study we investigate some of the pre-analytic considerations for isolating plasma fractions for the study of miRNA biomarkers. We find that measurement of circulating miRNA levels are frequently confounded by varying levels of cellular miRNAs of different hematopoietic origins. In order to assess the relative proportions of this cell-derived class, we have fractionated whole blood into plasma and its ensuing sub-fractions. Cellular miRNA signatures in cohorts of normal individuals are catalogued and the abundance and gender specific expression of bona fide circulating markers explored after calibrating the signal for this interfering class. A map of differentially expressed profiles is presented and the intrinsic variability of circulating miRNA species investigated in subsets of healthy males and females.

Show MeSH
Related in: MedlinePlus