Delayed processing of blood samples impairs the accuracy of mRNA-based biomarkers

Background: The transcriptome of peripheral white blood cells (PWBCs) contains valuable physiological information, thus making them a prime biological sample for investigating mRNA-based biomarkers. However, prolonged storage of whole blood samples can alter gene transcript abundance in PWBCs, compromising the results of biomarker discovery. Here, we designed an experiment to interrogate the impacts of delayed processing of whole blood samples on gene transcript abundance in PWBCs. We hypothesized that storing blood samples for 24 hours at 4°C would cause RNA degradation resulting in altered transcriptome profiles. Results: We produced RNA-sequencing data for 30 samples collected from five estrus synchronized heifers (Bos taurus). We quantified transcript abundance for 12,414 protein-coding genes in PWBCs. Analysis of parameters of RNA quality revealed no statistically significant differences (P>0.05) between samples collected from the jugular vein and coccygeal vein, as well as among samples processed after one, three, six, or eight hours. However, samples processed after 24 hours of storage had a lower RNA integrity number value (P=0.03) in comparison to those processed after one hour of storage. Next, we analyzed RNA-sequencing data between samples using those processed after one hour of storage as the baseline for comparison. Interestingly, evaluation of 3/5 prime bias revealed no differences between genes with lower transcript abundance in samples stored for 24 hours relative to one hour. In addition, sequencing coverage of transcripts was similar between samples from the 24-hour and one-hour groups. We identified four and 515 genes with differential transcript abundance in samples processed after storage for eight and 24 hours, respectively, relative to samples processed after one hour. Conclusions: The PWBCs respond to prolonged cold storage by increasing genes related to active chromatin compaction which in turn reduces gene transcription. This alteration in transcriptome profiles can impair the accuracy of mRNA-based biomarkers. Therefore, blood samples collected for mRNA-based biomarker discovery should be refrigerated immediately and processed within six hours post sampling.

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deconvSeq: deconvolution of cell mixture distribution in sequencing data

Bioinformatics ◽

10.1093/bioinformatics/btz444 ◽

2019 ◽

Vol 35 (24) ◽

pp. 5095-5102 ◽

Cited By ~ 10

Author(s):

Rose Du ◽

Vince Carey ◽

Scott T Weiss

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Whole Blood ◽

Bisulfite Sequencing ◽

Supplementary Information ◽

Tissue Type ◽

Cell Type ◽

Sequencing Data ◽

Blood Samples ◽

Whole Blood Samples

Abstract Motivation Although single-cell sequencing is becoming more widely available, many tissue samples such as intracranial aneurysms are both fibrous and minute, and therefore not easily dissociated into single cells. To account for the cell type heterogeneity in such tissues therefore requires a computational method. We present a computational deconvolution method, deconvSeq, for sequencing data (RNA and bisulfite) obtained from bulk tissue. This method can also be applied to single-cell RNA sequencing data. Results DeconvSeq utilizes a generalized linear model to model effects of tissue type on feature quantification, which is specific to the data structure of the sequencing type used. Estimated model coefficients can then be used to predict the cell type mixture within a tissue. Predicted cell type mixtures were validated against actual cell counts in whole blood samples. Using this method, we obtained a mean correlation of 0.998 (95% CI 0.995–0.999) from the RNA sequencing data of 35 whole blood samples and 0.95 (95% CI 0.91–0.98) from the reduced representation bisulfite sequencing data from 35 whole blood samples. Using symmetric balances to obtain the correlation between compositional parts, we found that the lowest correlation occurred for monocytes for both RNA and bisulfite sequencing. Comparison with other methods of decomposition such as deconRNAseq, CIBERSORT, MuSiC and EpiDISH showed that deconvSeq is able to achieve good prediction using mean correlation with far fewer genes or CpG sites in the signature set. Availability and implementation Software implementing deconvSeq is available at https://github.com/rosedu1/deconvSeq. Supplementary information Supplementary data are available at Bioinformatics online.

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Blood RNA Sequencing Confirms Upregulated BATF2 and FCGR1A Expression in Children with Autism Spectrum Disorder

10.21203/rs.3.rs-1113898/v1 ◽

2021 ◽

Author(s):

Irena Voinsky ◽

Yazeed Zoabi ◽

Noam Shomron ◽

Moria Harel ◽

Hanoch Cassuto ◽

...

Keyword(s):

Autism Spectrum Disorder ◽

Cancer Risk ◽

Rna Sequencing ◽

Whole Blood ◽

Autism Spectrum ◽

Spectrum Disorder ◽

Rna Seq ◽

Blood Samples ◽

Children With Asd ◽

Whole Blood Samples

Abstract Mutations in over 100 genes are implicated in autism spectrum disorder (ASD). DNA mutations and epigenomic modifications also contribute to ASD. Transcriptomics analysis of blood samples may offer clues for pathways dysregulated in ASD. To expand and validate published findings of RNA-sequencing (RNA-seq) studies, we performed RNA-seq of whole blood samples from a discovery cohort of eight children with ASD compared with nine age- and sex-matched neurotypical children. This revealed 10 genes with differential expression. Using real-time PCR, we compared whole blood samples from 35 children with ASD and 21 matched neurotypical children for the 10 dysregulated genes detected by RNA-seq. This revealed higher expression levels of the proinflammatory transcripts BATF2 and FCGR1A, and lower expression levels of the anti-inflammatory transcripts ISG15 and MT2A in the ASD compared to the control group. BATF2 and FCGR1A were recently reported as upregulated in blood samples of Japanese adults with ASD. Coupled with that publication, our findings support involvement of these genes in ASD phenotypes, independent of age and ethnicity. Upregulation of BATF2 and FCGR1A and downregulation of ISG15 and MT2A were reported to reduce cancer risk. Implications of the dysregulated genes for pro-inflammatory phenotypes, immunity, and cancer risk in ASD are discussed.

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PSXII-4 Impact of delayed processing time on total RNA quality and quantitation of transcript abundance

Journal of Animal Science ◽

10.1093/jas/skab235.456 ◽

2021 ◽

Vol 99 (Supplement_3) ◽

pp. 250-250

Author(s):

Chace Wilson ◽

Nicholas W Wege Dias ◽

Stefania Pancini ◽

Vitor R G Mercadante ◽

Fernando Biase

Keyword(s):

Processing Time ◽

White Blood Cells ◽

Transcript Abundance ◽

Rna Degradation ◽

Biological Information ◽

Gene Transcript ◽

Sample Collection ◽

Blood Samples ◽

Total Rna ◽

Rna Integrity

Abstract Critical biological information related to economically important traits is present in the blood transcriptome in cattle. Following a sample collection, however, RNA molecules containing this valuable information are prone to degradation affecting transcript abundance. Thus, proper sample handling and storage is essential when working with RNA. Here, we hypothesized that delayed time between sample collection and processing would lead to RNA degradation and alter gene transcript quantification. We aimed to determine the effect of delayed processing of whole blood on transcriptomic profiles in peripheral white blood cells (PWBC). We collected blood samples (10ml) in tubes containing anticoagulant (K2EDTA) from estrus synchronized beef heifers (n = 5). The tubes remained chilled on ice until processing time. From each heifer, we collected five samples from the jugular vein. We processed one sample from each animal within one hour of collection, and we delayed the processing time of the remaining samples to three, six, eight, and 24 hours post collection. We extracted total RNA from PWBCs, measured yield and assessed quality. We also quantified transcript abundance of 12,724 genes by RNA-sequencing. Samples processed 24 hours post collection had lower RNA integrity number (RIN) compared to samples processed withing one hour of collection (RIN24h=8.00±0.37, RIN1h=8.52±0.37, P = 0.03). There were four and 504 genes with differential transcript abundance (FDR< 0.05) when comparing eight and 24 hours of delayed processing with samples processed within one hour of collection, respectively. Notably, several genes had >1.4-fold greater transcript abundance when samples were stored on ice for eight or 24 hours. Our results indicate that RNA degradation and cellular activity of PWBCs have critical impact on transcript abundance when blood samples are stored for 24 hours under refrigeration. As the development of RNA-based biomarkers gain importance in cattle production systems, timely handling of samples is critical for accurate results.

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Bioinformatic removal of NUMT-associated variants in mitotiling next-generation sequencing data from whole blood samples

Electrophoresis ◽

10.1002/elps.201800135 ◽

2018 ◽

Vol 39 (21) ◽

pp. 2785-2797 ◽

Cited By ~ 19

Author(s):

Joseph David Ring ◽

Kimberly Sturk-Andreaggi ◽

Michelle Alyse Peck ◽

Charla Marshall

Keyword(s):

Next Generation Sequencing ◽

Whole Blood ◽

Next Generation Sequencing Data ◽

Next Generation ◽

Sequencing Data ◽

Blood Samples ◽

Whole Blood Samples ◽

Generation Sequencing

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RNA sequencing of the in vivohuman herpesvirus 6B transcriptome to identify targets for clinical assays distinguishing between latent and active infections

10.1101/397679 ◽

2018 ◽

Cited By ~ 1

Author(s):

Joshua A. Hill ◽

Minako Ikoma ◽

Danielle M. Zerr ◽

Ryan S. Basom ◽

Vikas Peddu ◽

...

Keyword(s):

Gene Expression ◽

Viral Infection ◽

Rna Sequencing ◽

Whole Blood ◽

Human Herpesvirus ◽

Viral Dna ◽

Blood Samples ◽

Whole Blood Samples

ABSTRACTHuman herpesvirus 6B (HHV-6B) DNA is frequently detected in human samples, especially after hematopoietic cell transplantation (HCT). Diagnostic assays distinguishing HHV-6B reactivation from latency are limited, and this has contributed to confusion in research and made the design of clinical approaches to diagnose and treat HHV-6-associated diseases challenging. We used RNA sequencing to characterize and compare the HHV-6B transcriptome in multiplein vivoandin vitrosample types, including 1) whole blood from HCT recipients with and without HHV-6B plasma viremia; 2) tumor tissue samples from subjects with large B cell lymphoma infected with HHV-6B; 3) lymphoblastoid cell lines from subjects with inherited chromosomally integrated HHV-6B or latent infection with HHV-6B; and 4) HHV-6B Z29 infected SupT1 CD4+ T cells. We demonstrated substantial overlap in the HHV-6B transcriptome observed inin vivoandin vitrosamples, although there was variability in the breadth and quantity of gene expression across samples. No HHV-6B transcripts were detected in whole blood samples from subjects without plasma HHV-6B viremia. The HHV-6B viral polymerase gene U38 was the only HHV-6B transcript detected in all RNA-seq data sets and was one of the most highly expressed genes. Using a novel reverse transcription PCR assay targeting HHV-6B U38, we identified U38 messenger RNA in all tested whole blood samples from patients with concurrent HHV-6B viremia, indicating its utility as a diagnostic assay for HHV-6B replication. This study demonstrates the feasibility of pathogen transcriptome analyses in HCT recipients to identify better targets for diagnostic, and potentially therapeutic, applications.IMPORTANCEInfection with human herpesvirus 6B (HHV-6B), a DNA virus, occurs early in life, results in chronic viral latency in diverse cell types, and affects the population at large. Additionally, HHV- 6B can integrate into germline chromosomes, resulting in individuals with viral DNA in every nucleated cell. Given that PCR to detect viral DNA is the mainstay for diagnosing HHV-6B infection, the characteristics of HHV-6B infection complicate efforts to distinguish between latent and active viral infection, particularly in immunocompromised patients who have frequent HHV- 6B reactivation. In this study, we used RNA sequencing to characterize the HHV-6B gene expression profile in multiple sample types, and our findings identified evidence-based targets for diagnostic tests that distinguish between latent and active viral infection.

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RNA Sequencing of theIn VivoHuman Herpesvirus 6B Transcriptome To Identify Targets for Clinical Assays Distinguishing between Latent and Active Infections

Journal of Virology ◽

10.1128/jvi.01419-18 ◽

2018 ◽

Vol 93 (3) ◽

Cited By ~ 4

Author(s):

Joshua A. Hill ◽

Minako Ikoma ◽

Danielle M. Zerr ◽

Ryan S. Basom ◽

Vikas Peddu ◽

...

Keyword(s):

Gene Expression ◽

Rna Sequencing ◽

Whole Blood ◽

Latent Infection ◽

Human Herpesvirus ◽

Rna Seq ◽

Viral Dna ◽

Blood Samples ◽

Multiple Sample ◽

Whole Blood Samples

ABSTRACTHuman herpesvirus 6B (HHV-6B) DNA is frequently detected in human samples. Diagnostic assays distinguishing HHV-6B reactivation from latency are limited. This has impaired strategies to diagnose and treat HHV-6B-associated diseases. We used RNA sequencing to characterize and compare the HHV-6B transcriptome in multiple sample types, including (i) whole blood from hematopoietic cell transplant (HCT) recipients with and without HHV-6B plasma viremia, (ii) tumor tissue samples from subjects with large B cell lymphoma infected with HHV-6B, (iii) lymphoblastoid cell lines (LCLs) from subjects with inherited chromosomally integrated HHV-6B or latent infection with HHV-6B, and (iv) HHV-6B Z29 infected SupT1 CD4+T cells. We demonstrated substantial overlap in the HHV-6B transcriptome observed inin vivoandin vitrosamples, although there was variability in the breadth and quantity of gene expression across samples. The HHV-6B viral polymerase gene U38 was the only HHV-6B transcript detected in all next-generation RNA sequencing (RNA-seq) data sets and was one of the most highly expressed genes. We developed a novel reverse transcription-PCR assay targeting HHV-6B U38, which identified U38 mRNA in all tested whole-blood samples from patients with concurrent HHV-6B viremia. No HHV-6B U38 transcripts were detected by RNA-seq or reverse transcription–real-time quantitative PCR (RT-qPCR) in whole-blood samples from subjects without HHV-6B plasma detection or from latently infected LCLs. A RT-qPCR assay for HHV-6B U38 may be useful to identify lytic HHV-6B infection in nonplasma samples and samples from individuals with inherited chromosomally integrated HHV-6B. This study also demonstrates the feasibility of transcriptomic analyses for HCT recipients.IMPORTANCEHuman herpesvirus 6B (HHV-6B) is a DNA virus that infects most children within the first few years of life. After primary infection, HHV-6B persists as a chronic, latent infection in many cell types. Additionally, HHV-6B can integrate into germ line chromosomes, resulting in individuals with viral DNA in every nucleated cell. Given that PCR to detect viral DNA is the mainstay for diagnosing HHV-6B infection, the characteristics of HHV-6B infection complicate efforts to distinguish between latent and active viral infection, particularly in immunocompromised patients who have frequent HHV-6B reactivation. In this study, we used RNA sequencing to characterize the HHV-6B gene expression profile in multiple sample types, and our findings identified evidence-based targets for diagnostic tests that distinguish between latent and active viral infection.

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