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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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Comparison of expression levels of circulating and cellular miRNAs in the CS and S1 fractions.(A) Intensity distributions from the highest expressed 20, 35, 50 or all 534 human miRNAs in the CS and S1 fractions after removal of contaminant features. P-values from paired Student's t-tests, contrasting the intensities for each pair of conditions are reported. (B) Intensity distributions from the highest expressed 20, 35, 50 or all 313 contaminant miRNAs in the CS and S1 fractions with p-values from paired t-tests measuring significance.
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pone-0020769-g004: Comparison of expression levels of circulating and cellular miRNAs in the CS and S1 fractions.(A) Intensity distributions from the highest expressed 20, 35, 50 or all 534 human miRNAs in the CS and S1 fractions after removal of contaminant features. P-values from paired Student's t-tests, contrasting the intensities for each pair of conditions are reported. (B) Intensity distributions from the highest expressed 20, 35, 50 or all 313 contaminant miRNAs in the CS and S1 fractions with p-values from paired t-tests measuring significance.

Mentions: Since the process of sub-fractionation may impact both the integrity and abundance of miRNAs, we wanted to ensure that markers that were conserved between the CS and the S1 fractions did not vary in their expression levels. In order to test this, categories of miRNAs that are common to both these fractions were selected after filtering for contaminants and stratified into different intensity bins consisting of the top 20, 35, 50 or all of the 534 miRNAs (total number of features that remain after removal of 313 contaminant miRNAs). We observe no significant difference in the expression levels for any of these miRNA classes in either of the supernatant fractions (two sided Student's t–test, with p-values ranging from 0.053 to 0.596) (Fig. 4A). This indicates that most miRNA species remain intact through the separation process from CS to S1. In addition, since the isolation process is predicted to remove contaminant miRNAs we anticipate higher proportions of cellular RNAs in the CS fraction compare to the S1 class. To directly test this we similarly compared expression levels of cellular RNAs stratified into different intensity tiers between the two plasma derived fractions. For all miRNA strata compared, we observe a statistically significant down regulation in abundances for cellular miRNAs in the S1 class with a p-value of <2.3e−12 (Fig. 4B). This indicates a clarification of these RNAs from the CS to the subsequently derived S1 class as a consequence of the fractionation process. Taken together these result suggests that isolation of the S1 class from the CS minimizes the levels of contaminating cellular RNA, while preserving the expression of circulating miRNA species.


Impact of cellular miRNAs on circulating miRNA biomarker signatures.

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

Comparison of expression levels of circulating and cellular miRNAs in the CS and S1 fractions.(A) Intensity distributions from the highest expressed 20, 35, 50 or all 534 human miRNAs in the CS and S1 fractions after removal of contaminant features. P-values from paired Student's t-tests, contrasting the intensities for each pair of conditions are reported. (B) Intensity distributions from the highest expressed 20, 35, 50 or all 313 contaminant miRNAs in the CS and S1 fractions with p-values from paired t-tests measuring significance.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3117799&req=5

pone-0020769-g004: Comparison of expression levels of circulating and cellular miRNAs in the CS and S1 fractions.(A) Intensity distributions from the highest expressed 20, 35, 50 or all 534 human miRNAs in the CS and S1 fractions after removal of contaminant features. P-values from paired Student's t-tests, contrasting the intensities for each pair of conditions are reported. (B) Intensity distributions from the highest expressed 20, 35, 50 or all 313 contaminant miRNAs in the CS and S1 fractions with p-values from paired t-tests measuring significance.
Mentions: Since the process of sub-fractionation may impact both the integrity and abundance of miRNAs, we wanted to ensure that markers that were conserved between the CS and the S1 fractions did not vary in their expression levels. In order to test this, categories of miRNAs that are common to both these fractions were selected after filtering for contaminants and stratified into different intensity bins consisting of the top 20, 35, 50 or all of the 534 miRNAs (total number of features that remain after removal of 313 contaminant miRNAs). We observe no significant difference in the expression levels for any of these miRNA classes in either of the supernatant fractions (two sided Student's t–test, with p-values ranging from 0.053 to 0.596) (Fig. 4A). This indicates that most miRNA species remain intact through the separation process from CS to S1. In addition, since the isolation process is predicted to remove contaminant miRNAs we anticipate higher proportions of cellular RNAs in the CS fraction compare to the S1 class. To directly test this we similarly compared expression levels of cellular RNAs stratified into different intensity tiers between the two plasma derived fractions. For all miRNA strata compared, we observe a statistically significant down regulation in abundances for cellular miRNAs in the S1 class with a p-value of <2.3e−12 (Fig. 4B). This indicates a clarification of these RNAs from the CS to the subsequently derived S1 class as a consequence of the fractionation process. Taken together these result suggests that isolation of the S1 class from the CS minimizes the levels of contaminating cellular RNA, while preserving the expression of circulating miRNA 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