Globin mRNA reduction for whole-blood transcriptome sequencing

Krjutškov, Kaarel; Koel, Mariann; Roost, Anne Mari; Katayama, Shintaro; Einarsdottir, Elisabet; Jouhilahti, Eeva-Mari; Söderhäll, Cilla; Jaakma, Ülle; Plaas, Mario; Vesterlund, Liselotte; Lohi, Hannes; Salumets, Andres; Kere, Juha

doi:10.1038/srep31584

Cited by 40 publications

(39 citation statements)

References 22 publications

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“…The GB applied in QuantSeq library construction successfully reduced the proportions of HBA and HBB reads and showed optimum efficiency with concentrations C1 and C2 of the globin blocker compared to higher concentrations ( Figure 2). The GB more than doubled the proportion of reads that mapped to non-globin genes and this led to an increase in the number of detected genes (Figure 3), which agrees with previous studies on globin depletion in RNAseq [10,[12][13][14]. There were, however, some genes which were detected in the NGB samples but not in the corresponding GB samples, but these genes did not show sequence similarity with HBA or HBB.…”

Section: Discussionsupporting

confidence: 89%

“…Previous studies have shown that globin depletion methods in RNA sequencing such as GLOBINclear TM (ThermoFisher), the modified GR protocol (Affymetrix), and GlobinLock are effective in reducing globin mRNA in blood [10,[12][13][14]. However, these studies used a small number of samples and could not validate that the globin depletion method worked uniformly across samples.…”

Section: Discussionmentioning

confidence: 98%

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The effects of a globin blocker on the resolution of 3’mRNA sequencing data in porcine blood

Lim

Moll²,

Vitkovska³

et al. 2019

Preprint

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Background : Gene expression profiling in blood is a potential source of biomarkers to evaluate or predict phenotypic differences between pigs but is expensive and inefficient because of the high abundance of globin mRNA in porcine blood. These limitations can be overcome by the use of 3’mRNA sequencing combined with a method to deplete or block the processing of globin mRNA prior to or during library construction. Here, we validated the effectiveness of a novel specific globin blocker (GB) that is included in the library preparation step of 3’mRNA sequencing. Results : Four concentrations of the GB were applied to RNA samples from two pigs. The GB significantly reduced the proportion of globin reads ( P = 0.005) and increased the number of detectable non-globin genes. The highest evaluated concentration (C1) of the GB resulted in the largest reduction of globin reads (from 56.4 to 10.1%). The second highest concentration C2, showed very similar globin depletion rates (12 %) as C1 but a better correlation of the expression of non-globin genes between GB and non-GB ( r = 0.98), and allowed the expression of an additional 1,295 non-globin genes to be detected. Concentration C2 was applied in the rest of the study. The distribution of the percentage of globin reads for non-GB (n=184) and GB (n=189) samples clearly showed the effects of the GB on reducing globin reads, in particular for HBB . The proportion of globin reads that remained in GB samples was found to be positively correlated with reticulocyte count of the blood sample ( P < 0.001). Conclusions : The GB for 3’mRNA sequencing is a useful tool in the quantification of whole gene expression profiles in porcine blood. The GB reduced the proportion of globin reads, thereby increasing the efficiency of sequencing non-globin mRNA. The evaluated GB method has as additional advantage that it does not require an additional step prior to or during library creation.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 98%

The effects of a globin blocker on the resolution of 3’mRNA sequencing data in porcine blood

Lim

Moll²,

Vitkovska³

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…On the other hand, though, one of the key challenges of the analysis of whole blood transcriptome is the overabundance of globin mRNA, which may decrease the ability to detect transcripts with low expression levels. Several experimental protocols of globin depletion have been proposed, which however significantly reduce the amount of extracted RNA following depletion [15] and compromise the quality of the nucleic acid [16]. Any loss of RNA caused by depletion might affect the sequencing data, particularly for samples with a limited amount of starting material.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Transcriptomic Regulations behind Metabolic Syndrome in Obese and Lean Subjects

Paczkowska-Abdulsalam

Niemira

Bielska

et al. 2020

IJMS

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Multiple mechanisms have been suggested to confer to the pathophysiology of metabolic syndrome (MetS), however despite great interest from the scientific community, the exact contribution of each of MetS risk factors still remains unclear. The present study aimed to investigate molecular signatures in peripheral blood of individuals affected by MetS and different degrees of obesity. Metabolic health of 1204 individuals from 1000PLUS cohort was assessed, and 32 subjects were recruited to four study groups: MetS lean, MetS obese, “healthy obese”, and healthy lean. Whole-blood transcriptome next generation sequencing with functional data analysis were carried out. MetS obese and MetS lean study participants showed the upregulation of genes involved in inflammation and coagulation processes: granulocyte adhesion and diapedesis (p < 0.0001, p = 0.0063), prothrombin activation pathway (p = 0.0032, p = 0.0091), coagulation system (p = 0.0010, p = 0.0155). The results for “healthy obese” indicate enrichment in molecules associated with protein synthesis (p < 0.0001), mitochondrial dysfunction (p < 0.0001), and oxidative phosphorylation (p < 0.0001). Our results suggest that MetS is related to the state of inflammation and vascular system changes independent of excess body weight. Furthermore, “healthy obese”, despite not fulfilling the criteria for MetS diagnosis, seems to display an intermediate state with a lower degree of metabolic abnormalities, before they proceed to a full blown MetS.

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“…The resulting amounts and purity of reticulocytes were sufficient for the efficient, high quality RNA isolation required in the NGS‐based transcriptome analyses (Table and Figure ). It should be noted that in the case of reticulocytes obtained from healthy individuals (where extensive hemolysis of red blood cells is not observed), the addition of globin reduction step may be considered, which can enrich data obtained from next generation sequencing‐based expression profiling …”

Section: Discussionmentioning

confidence: 99%

Efficient method for isolation of reticulocyte RNA from healthy individuals and hemolytic anaemia patients

Skulski

Bartoszewski

Majkowski

et al. 2018

J Cellular Molecular Medi

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Despite enormous progress and development of high‐throughput methods in genome‐wide mRNA analyses, data on the erythroid transcriptome are still limited, even though they could be useful in medical diagnostics and personalized therapy as well as in research on normal and pathological erythroid maturation. Although obtaining normal and pathological reticulocyte transcriptome profiles should contribute greatly to our understanding of the molecular bases of terminal erythroid differentiation as well as the mechanisms of the hematological diseases, a basic limitation of these studies is the difficulty of efficient reticulocyte RNA isolation from human peripheral blood. The restricted number of possible parallel experiments primarily concern healthy individuals with the lowest number of reticulocytes in the peripheral blood and a low RNA content. In the present study, an efficient method for reticulocyte RNA isolation from healthy individuals and hemolytic anaemia patients is presented. The procedure includes leukofiltration, Ficoll‐Paque gradient centrifugation, Percoll gradient centrifugation, and negative (CD45 and CD61) immunomagnetic separation. This relatively fast and simple four‐stage method was successfully applied to obtain a reticulocyte‐rich population from healthy subjects, which was used to efficiently isolate the high‐quality RNA essential for successful NGS‐based transcriptome analysis.

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Globin mRNA reduction for whole-blood transcriptome sequencing

Cited by 40 publications

References 22 publications

The effects of a globin blocker on the resolution of 3’mRNA sequencing data in porcine blood

The effects of a globin blocker on the resolution of 3’mRNA sequencing data in porcine blood

Evaluation of Transcriptomic Regulations behind Metabolic Syndrome in Obese and Lean Subjects

Efficient method for isolation of reticulocyte RNA from healthy individuals and hemolytic anaemia patients

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