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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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Fractionation Workflow.Separation of whole blood into distinct fractions: WBC, RBC, Leukocytes and CS, S1, S2, P1, P2 through differential centrifugation. Total RNA was extracted from each fraction and hybridized to miRNA arrays.
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pone-0020769-g001: Fractionation Workflow.Separation of whole blood into distinct fractions: WBC, RBC, Leukocytes and CS, S1, S2, P1, P2 through differential centrifugation. Total RNA was extracted from each fraction and hybridized to miRNA arrays.

Mentions: Conventional protocols for isolation of plasma (the non-cellular component of blood that remains after removal of cells by centrifugation) involves variable combinations of individual or multiple low/high speed centrifugation steps. In order to assess the contribution of these differential spin steps in the removal of cellular material, we sought to fractionate whole blood from 17 healthy male Caucasian donors (Table S1) through successive centrifugation rounds and investigate the proportion of cell free and cellular miRNAs at each stage (Fig. 1). Samples were initially segregated at a low speed spin to generate plasma (Cloudy Supernatant) along with three cellular fractions: (1) Red Blood Cells (RBCs), (2) the Buffy Coat consisting of platelets and White Blood Cells (WBCs) and (3) pure populations of leukocytes isolated by sub-fractionation of blood through Ficoll-Hypaque gradients (Leukocytes). Since these populations represent the major cellular constituents of blood, we hypothesize them to be the primary contributor of cellular material through either cell carryover or lysis during blood fractionation and collectively define them as contaminant miRNAs. We further segregated the plasma derived cloudy supernatant layer through increasing centrifugal forces into supernatant (S1 and S2) and pellet (P1 and P2) fractions. The supernatant fractions are representative of primarily cell-free circulating RNAs with the pellet fractions characterizing contaminating cellular particles. Amongst the pellet class – the P1 and P2 fractions are distinct in both size and granularity of the isolates. A prominent precipitate is distinguished in the P1 fraction while the P2 fraction has inconsequential cellular particles. All eight fractions (Fig. 1) were extracted using Trizol-LS (Invitrogen) and the mirVANA filters (Ambion) and subsequently hybridized to the Affymetrix miRNA arrays. Integrity of extracted RNA was evaluated by Bioanalyzer or Polyacrylamide Gel Electrophoresis and for all fractions samples were found to be of robust quality (Fig. S1). On analysis of the data, we observe distinct gradients in intensity distributions in all of the 5 plasma derived classes compared to the contaminant miRNAs (Fig. 2A). Fractions isolated with minimal centrifugation (Cloudy supernatant) or hypothesized to be contaminant rich (P1 and P2) display a greater magnitude of signal intensity compared to supernatant fractions of S1 and S2. Taken together these distributions indicate the presence of miRNAs of distinct abundances in the cellular and circulating class and highlights the prevalence of low-abundant species in plasma fractions clarified through successive purification steps.


Impact of cellular miRNAs on circulating miRNA biomarker signatures.

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

Fractionation Workflow.Separation of whole blood into distinct fractions: WBC, RBC, Leukocytes and CS, S1, S2, P1, P2 through differential centrifugation. Total RNA was extracted from each fraction and hybridized to miRNA arrays.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020769-g001: Fractionation Workflow.Separation of whole blood into distinct fractions: WBC, RBC, Leukocytes and CS, S1, S2, P1, P2 through differential centrifugation. Total RNA was extracted from each fraction and hybridized to miRNA arrays.
Mentions: Conventional protocols for isolation of plasma (the non-cellular component of blood that remains after removal of cells by centrifugation) involves variable combinations of individual or multiple low/high speed centrifugation steps. In order to assess the contribution of these differential spin steps in the removal of cellular material, we sought to fractionate whole blood from 17 healthy male Caucasian donors (Table S1) through successive centrifugation rounds and investigate the proportion of cell free and cellular miRNAs at each stage (Fig. 1). Samples were initially segregated at a low speed spin to generate plasma (Cloudy Supernatant) along with three cellular fractions: (1) Red Blood Cells (RBCs), (2) the Buffy Coat consisting of platelets and White Blood Cells (WBCs) and (3) pure populations of leukocytes isolated by sub-fractionation of blood through Ficoll-Hypaque gradients (Leukocytes). Since these populations represent the major cellular constituents of blood, we hypothesize them to be the primary contributor of cellular material through either cell carryover or lysis during blood fractionation and collectively define them as contaminant miRNAs. We further segregated the plasma derived cloudy supernatant layer through increasing centrifugal forces into supernatant (S1 and S2) and pellet (P1 and P2) fractions. The supernatant fractions are representative of primarily cell-free circulating RNAs with the pellet fractions characterizing contaminating cellular particles. Amongst the pellet class – the P1 and P2 fractions are distinct in both size and granularity of the isolates. A prominent precipitate is distinguished in the P1 fraction while the P2 fraction has inconsequential cellular particles. All eight fractions (Fig. 1) were extracted using Trizol-LS (Invitrogen) and the mirVANA filters (Ambion) and subsequently hybridized to the Affymetrix miRNA arrays. Integrity of extracted RNA was evaluated by Bioanalyzer or Polyacrylamide Gel Electrophoresis and for all fractions samples were found to be of robust quality (Fig. S1). On analysis of the data, we observe distinct gradients in intensity distributions in all of the 5 plasma derived classes compared to the contaminant miRNAs (Fig. 2A). Fractions isolated with minimal centrifugation (Cloudy supernatant) or hypothesized to be contaminant rich (P1 and P2) display a greater magnitude of signal intensity compared to supernatant fractions of S1 and S2. Taken together these distributions indicate the presence of miRNAs of distinct abundances in the cellular and circulating class and highlights the prevalence of low-abundant species in plasma fractions clarified through successive purification steps.

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