Dataset Information

Proteomic Profiling of HEK293-F cell and human peripheral blood neutrophil proteins extracted by conventional direct extraction and phenol-guanidinium isothiocyanate (P/GTC)-based extraction methods

ABSTRACT: To evaluate the performance of the phenol-guanidinium isothiocyanate (P/GTC)-based extraction method in proteomic analysis, we performed proteome analysis from HEK293-F cell and human peripheral blood neutrophil proteins extracted by the P/GTC-based extraction and conventional direct extraction method. Furthermore, the P/GTC extracted proteins were analyzed by 2D-LC-MS/MS.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yusuke Kawashima

PROVIDER: PXD011487 | JPOST Repository | Mon Oct 28 00:00:00 GMT 2019

REPOSITORIES: jPOST

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Publications

Proteogenomic Analyses of Cellular Lysates Using a Phenol-Guanidinium Thiocyanate Reagent.

Kawashima Yusuke Y Miyata Jun J Watanabe Takashi T Shioya Juri J Arita Makoto M Ohara Osamu O

Journal of proteome research 20181112 1

Protein and RNA profiles are highly informative when generating a comprehensive understanding of the dynamics of complex biological systems. Quantitative correlations between protein and RNA profiles are not always high, yet simultaneous acquisition of both profiles remains challenging, in part because of the limited availability of samples and the inconvenience of separately preparing protein and RNA fractions. In a previous study, protein, DNA, and RNA fractions were simultaneously prepared fr ...[more]

PMID: 30394753

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Project description:Gene expression profiling using blood samples is a valuable tool for biomarker discovery in clinical studies. Different whole blood RNA collection and processing methods are highly variable and might confound comparisons of results across studies. The main aim of the study was to compare how blood storage, extraction methodologies, and the blood component itself may influence gene expression profiling where samples were collected in triplicate from five healthy donors. Whole blood was collected in RNAGard™, PAXgene™ as well as collection tubes with anticoagulant such as dipotassium ethylenediaminetetraacetic acid (K2EDTA) and acid citrate dextrose solution A (ACD-A). Peripheral blood mononuclear cells (PBMCs) were separated using sodium citrate cell preparation tubes (CPT), FICOLL™, immunomagnetic and Leukolock™ methods. The Leukolock™, K2EDTA and ACD-A blood tubes were shipped overnight using cold conditions and rest of the methods were immediately frozen with or without pre-processing. The RNA was isolated from whole blood and peripheral blood mononuclear cells (PBMCs) with a total of 10 different experimental conditions employing several widely utilized RNA isolation methods. The RNA quality as assessed by RNA integrity Number (RIN) showed that all PBMC procedures had the highest RIN values when processed and stabilized in TRIzol before RNA extraction. Initial data analysis showed that human blood stored at 4°C overnight performed equally well when compared to frozen stabilized blood. Comparisons within and across donor/method replicates showed signal to noise patterns which were not captured by RIN value alone. Our results provide some new insights into RNA isolation from blood samples, how the RNA isolation quality and apparent gene expression is influenced by the choice of storage, handling, and methodologies, and how careful consideration is necessary to avoid bias resulting from downstream processing. Better characterization of the effects of collection method idiosyncrasies will facilitate further research into understanding the effect of gene expression on variability in human sample types.

			Action	DRS
	HEK293_Direct_1.wiff	Wiff
	HEK293_Direct_1.wiff.scan	Wiff
	HEK293_Direct_2.wiff	Wiff
	HEK293_Direct_2.wiff.scan	Wiff
	HEK293_Direct_3.wiff	Wiff