Addition of β-Mercaptoethanol Is a Prerequisite for High-Quality RNA Isolation Using QIAsymphony Technology as Demonstrated by Detection of Molecular Aberrations in Hematologic Malignancies

Luytgaarde, Sonja C P A M van der Poel-van de; Geertsma-Kleinekoort, W M C; Goudswaard, Chantal; Hogenbirk-Hupkes, Pauline E; Hoven-Beijen, M Antoinette van; Werf, Marloes van de; Chu, Isabel W. T.; Kapel, J. Van; Valk, Peter J.M.

doi:10.1089/gtmb.2012.0448

Cited by 5 publications

(4 citation statements)

References 12 publications

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“…Although DTT is less pungent and toxic than β-ME, researchers are reluctant to use it because it is usually not specified in RNA extraction protocols and there is no published data evaluating its efficacy in RNA extractions. The only salient publication established that RNA extracted from leukocytes was less degraded when β-ME was included in the lysis buffer than when it was not (5). We therefore ask whether that reducing agent has to be β-ME, or whether it can be judiciously replaced with DTT.…”

Section: Introductionmentioning

confidence: 99%

Replacing β-mercaptoethanol in RNA extractions

Mommaerts

Sánchez

Betsou

et al. 2015

Analytical Biochemistry

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Section: Introductionmentioning

confidence: 99%

Replacing β-mercaptoethanol in RNA extractions

Mommaerts

Sánchez

Betsou

et al. 2015

Analytical Biochemistry

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“…Gaps in RNA integrity may depend primarily on the composition of the three kits. In kit 1, β-mercaptoethanol (β-ME), a well-known reducing agent that irreversibly denatures RNase by reducing disulfide bonds and destroying the native conformation ( van der Poel-van de Luytgaarde et al, 2013 ), was added to the leukocyte lysis buffer to eliminate the RNase. In kit 2, cells were lysed by guanidinium thiocyanate–phenol, which is also used in the TRIzol kit and can prevent the activity of RNA enzymes by denaturing them to yield undegraded RNA ( Chomczynski and Sacchi, 1987 ).…”

Section: Resultsmentioning

confidence: 99%

Improper preanalytical processes on peripheral blood compromise RNA quality and skew the transcriptional readouts of mRNA and LncRNA

Dong

et al. 2023

Front. Genet.

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Genetic and epigenetic reprogramming caused by disease states in other tissues is always systemically reflected in peripheral blood leukocytes (PBLs). Accurate transcriptional readouts of Messenger RNA (mRNA) and Long non-coding RNA (lncRNA) in peripheral blood leukocytes are fundamental for disease-related study, diagnosis and treatment. However, little is known about the impact of preanalytical variables on RNA quality and downstream messenger RNA and Long non-coding RNA readouts. In this study, we explored the impact of RNA extraction kits and timing of blood placement on peripheral blood leukocyte-derived RNA quality. A novel enhanced evaluation system including RNA yields, purity, RNA integrity number (RIN) values and β-actin copies was employed to more sensitively identify RNA quality differences. The expression levels of informative mRNAs and Long non-coding RNAs in patients with chronic obstructive pulmonary disease (COPD) or triple-negative breast cancer (TNBC) were measured by Quantitative reverse transcription polymerase chain reaction (qRT–PCR) to investigate the impact of RNA quality on transcriptional readouts. Our results showed that the quality of RNA extracted by different kits varies greatly, and commercial kits should be evaluated and managed before batch RNA extraction. In addition, the quality of extracted RNA was highly correlated with the timing of blood placement, and the copy number of β-actin was significantly decreased after leaving blood at RT over 12 h. More importantly, compromised RNA leads to skewed transcriptional readouts of informative mRNAs and Long non-coding RNAs in patients with chronic obstructive pulmonary disease or triple-negative breast cancer. These findings have significant implications for peripheral blood leukocyte-derived RNA quality management and suggest that quality control is necessary prior to the analysis of patient messenger RNA and Long non-coding RNA expression.

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“…Although Buffer RLT is a commercial lysis buffer which has been used in different protocols and kits for the lysis of cells and tissues prior to RNA extraction 33–35 , its effectiveness as a differential lysis buffer for human and fungal cells has not been studied yet.…”

Section: Discussionmentioning

confidence: 99%

Nucleic acids enrichment of fungal pathogens to study host-pathogen interactions

Rodríguez

Guillemyn

Coucke

et al. 2019

Sci Rep

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Fungal infections, ranging from superficial to life-threatening infections, represent a major public health problem that affects 25% of the worldwide population. In this context, the study of hostpathogen interactions within the host is crucial to advance antifungal therapy. However, since fungal cells are usually outnumbered by host cells, the fungal transcriptome frequently remains uncovered. We compared three different methods to selectively lyse human cells from in vitro mixes, composed of Candida cells and peripheral blood mononuclear cells. In order to prevent transcriptional modification, the mixes were stored in RNAlater. We evaluated the enrichment of fungal cells through cell counting using microscopy and aimed to further enrich fungal nucleic acids by centrifugation and by reducing contaminant nucleic acids from the host. We verified the enrichment of fungal DNA and RNA through qPCR and RT-qPCR respectively and confirmed that the resulting RNA has high integrity scores, suitable for downstream applications. The enrichment method provided here, i.e., lysis with Buffer RLT followed by centrifugation, may contribute to increase the proportion of nucleic acids from fungi in clinical samples, thus promoting more comprehensive analysis of fungal transcriptional profiles. Although we focused on C. albicans, the enrichment may be applicable to other fungal pathogens.

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Addition of β-Mercaptoethanol Is a Prerequisite for High-Quality RNA Isolation Using QIAsymphony Technology as Demonstrated by Detection of Molecular Aberrations in Hematologic Malignancies

Cited by 5 publications

References 12 publications

Replacing β-mercaptoethanol in RNA extractions

Replacing β-mercaptoethanol in RNA extractions

Improper preanalytical processes on peripheral blood compromise RNA quality and skew the transcriptional readouts of mRNA and LncRNA

Nucleic acids enrichment of fungal pathogens to study host-pathogen interactions

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