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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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Variability of circulating miRNA expression levels in normal cohorts of male and female individuals.(A) Box plot of intensity distributions of 140 features common to both circulation and in contaminants (+S/+L) or 47 features specific only to circulation (+S/−L). The black bar represents the median of each distribution. The open circles represent the outliers. (B) Analysis of Coefficient of Variance of these two categories. P-value from two-sided Student's t-test measuring tests of significance is reported.
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pone-0020769-g006: Variability of circulating miRNA expression levels in normal cohorts of male and female individuals.(A) Box plot of intensity distributions of 140 features common to both circulation and in contaminants (+S/+L) or 47 features specific only to circulation (+S/−L). The black bar represents the median of each distribution. The open circles represent the outliers. (B) Analysis of Coefficient of Variance of these two categories. P-value from two-sided Student's t-test measuring tests of significance is reported.

Mentions: The intrinsic variability in expression of a biomarker is a critical determinant for understanding both its normal behavior as well as assessing diseased induced changes. To explore the expression characteristics of circulating miRNAs, we investigated both the intensity and variability of expression of markers isolated from the S1 fraction in cohorts of normal individuals. Samples extracted from 8 male and 10 female Caucasian donors were background adjusted, quantile normalized and the summarized intensities for all microRNA and background probes analyzed (Table S1 and Fig. S2). In order to ensure that markers displaying a wide range of expression values were included in this analysis, individual miRNAs were selected upon presence/absence calls and categorized based on a 50% detection threshold amongst the 18 individuals. Two categories of miRNAs were defined in the S1 fraction: (1) 140 miRNAs present in both circulation (designated as S) and those that map cumulatively to the contaminant profile derived from WBC, RBC and Leukocytes (designated as L): (+S/+L) and (2) 47 miRNAs present in circulation only: (+S/−L) (Table S3, Fig. S3). The variability in each class was assessed through Coefficient of Variance (CV) estimates. We observe a statistically significant reduction in both intensities (p-value of 2.2e-16 from two-sided Student's t-test) (Fig. 6A) and CV measurements (p-value of 0.023) (Fig. 6B) in the circulating class (+S/−L) compared to miRNAs that are co-detected both in circulation and contaminants (+S/+L). This result demonstrates that the variability in expression of circulating miRNA species in a population is reduced after removal of signatures originating from contaminating cellular miRNA profiles. Furthermore, examination of the 47 circulating miRNAs reveals greater than two-fold dynamic range of intensity for this class (Fig. S3) and harbors candidates that bear evidence of expression derived from tissues of non-hematopoietic origin. Specifically we find hsa-miR-122 to be the highest expressing circulating miRNA in our current dataset with tissue specific expression derived from the liver (Fig. S3) [21], [22]. Our analysis therefore clearly distinguishes the effects on classification and variability that arise due to varying levels of cell-derived miRNAs in samples and underscores the importance of isolation practices for the study of circulating species.


Impact of cellular miRNAs on circulating miRNA biomarker signatures.

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

Variability of circulating miRNA expression levels in normal cohorts of male and female individuals.(A) Box plot of intensity distributions of 140 features common to both circulation and in contaminants (+S/+L) or 47 features specific only to circulation (+S/−L). The black bar represents the median of each distribution. The open circles represent the outliers. (B) Analysis of Coefficient of Variance of these two categories. P-value from two-sided Student's t-test measuring tests of significance is reported.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3117799&req=5

pone-0020769-g006: Variability of circulating miRNA expression levels in normal cohorts of male and female individuals.(A) Box plot of intensity distributions of 140 features common to both circulation and in contaminants (+S/+L) or 47 features specific only to circulation (+S/−L). The black bar represents the median of each distribution. The open circles represent the outliers. (B) Analysis of Coefficient of Variance of these two categories. P-value from two-sided Student's t-test measuring tests of significance is reported.
Mentions: The intrinsic variability in expression of a biomarker is a critical determinant for understanding both its normal behavior as well as assessing diseased induced changes. To explore the expression characteristics of circulating miRNAs, we investigated both the intensity and variability of expression of markers isolated from the S1 fraction in cohorts of normal individuals. Samples extracted from 8 male and 10 female Caucasian donors were background adjusted, quantile normalized and the summarized intensities for all microRNA and background probes analyzed (Table S1 and Fig. S2). In order to ensure that markers displaying a wide range of expression values were included in this analysis, individual miRNAs were selected upon presence/absence calls and categorized based on a 50% detection threshold amongst the 18 individuals. Two categories of miRNAs were defined in the S1 fraction: (1) 140 miRNAs present in both circulation (designated as S) and those that map cumulatively to the contaminant profile derived from WBC, RBC and Leukocytes (designated as L): (+S/+L) and (2) 47 miRNAs present in circulation only: (+S/−L) (Table S3, Fig. S3). The variability in each class was assessed through Coefficient of Variance (CV) estimates. We observe a statistically significant reduction in both intensities (p-value of 2.2e-16 from two-sided Student's t-test) (Fig. 6A) and CV measurements (p-value of 0.023) (Fig. 6B) in the circulating class (+S/−L) compared to miRNAs that are co-detected both in circulation and contaminants (+S/+L). This result demonstrates that the variability in expression of circulating miRNA species in a population is reduced after removal of signatures originating from contaminating cellular miRNA profiles. Furthermore, examination of the 47 circulating miRNAs reveals greater than two-fold dynamic range of intensity for this class (Fig. S3) and harbors candidates that bear evidence of expression derived from tissues of non-hematopoietic origin. Specifically we find hsa-miR-122 to be the highest expressing circulating miRNA in our current dataset with tissue specific expression derived from the liver (Fig. S3) [21], [22]. Our analysis therefore clearly distinguishes the effects on classification and variability that arise due to varying levels of cell-derived miRNAs in samples and underscores the importance of isolation practices for the study of circulating species.

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