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Blood Handling and Leukocyte Isolation Methods Impact the Global Transcriptome of Immune Cells

ABSTRACT:

In order to characterize the effects of upstream sample handling on the transcriptome of isolated leukocyte populations, we simulated various sample handling methods on whole blood prior to leukocyte isolation for low-input RNA-sequencing.

PROVIDER: phs001563 | dbGaP |

REPOSITORIES: dbGaP

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Project description:Single cell and low-input cell proteomics to measure cellular heterogeneity remains experimental and requires further refinement for routine application. This study aimed to build on previous protocols to assess the utility of targeted proteomics for measuring heterogeneity in red blood cells (RBCs). Pioneering developments, nanodroplet processing in one-pot for trace samples (nanoPOTS) and automated preparation in one-pot for trace samples (autoPOTS), allow single cell proteomics on proteins with copy numbers <106. Here, the latter workflow was further simplified to eliminate robotic liquid handling instrument and modified (10-port) nano-LC switching valve, required for online solid phase extraction. When combined with isotope labeled synthetic peptide standards for targeted quantitative isotope dilution parallel reaction monitoring (PRM) LC-MS (IDMS-PRM), protein quantification was completed in 2.5 h from sample preparation to data analysis. To assess the utility of the protocol for measuring (protein) cellular heterogeneity, an IDMS-PRM assay was developed to measure endogenous N-terminal proteolytic peptide of hemoglobin beta subunit and it’s N-terminal valine carboxymethyl (CMV) adduct, which were quantified in single digit red blood cells (RBCs), isolated via limiting-dilution (LD). Substituting limiting dilution with a more sophisticated isolation strategy, achieved with the CellenONE single cell dispenser, the endogenous concentration of the N-terminal proteolytic peptide of hemoglobin beta subunit, and by inference the hemoglobin tetramer, was measured in single red blood cells (540-660 amol/RBC) which was comparable to the calculated SI reference range for mean corpuscular hemoglobin (390-540 amol/RBC). Moreover, it was observed repeated measures using low input cell numbers (5-25 RBC) could be used as an alternative and less rigorous strategy (compared to single RBC analysis) to measure protein heterogeneity. This heterogeneity was observed when %CMV was repeatedly measured using low input RBCs (5-25 RBCs) in specimens obtained from N = 10 individuals with clinically measured HbA1c values. In conclusion, a rapid and relatively simplified protocol has been developed for targeted quantification of proteins to measure red blood cell heterogeneity.

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Blood Handling and Leukocyte Isolation Methods Impact the Global Transcriptome of Immune Cells

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