Replacing β-mercaptoethanol in RNA extractions

Mommaerts, Kathleen; Sánchez, I. H.; Betsou, Fay; Mathieson, William

doi:10.1016/j.ab.2015.03.027

Cited by 27 publications

(25 citation statements)

References 7 publications

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“…Since one of the priorities of the development of the method was the refusal to use toxic reagents, the widely used β-mercaptoethanol was replaced by dithiothreitol, which is practically odorless, resulting in no significant difference. Similar result is described earlier [ 3 ].…”

Section: Resultssupporting

confidence: 92%

One-phase phenol-free method for microRNA isolation from blood plasma

et al. 2018

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MicroRNA extraction is an essential procedure when discovering MicroRNA-based biomarkers and approaches. Here we describe a new method for microRNA isolation from human blood plasma, based on isopropanol precipitation from the one-phase lysate. We demonstrate that the use of more than four volumes of lysis buffer based on 5 M guanidine isothiocyanate prevents the formation of large, viscous, and hardly soluble precipitate. Applying widely used linear polyacrylamide (LPAA) as the only precipitating agent proved ineffective. At the same time, adding poly(A)RNA or tRNA with LPAA significantly increased the amount of microRNA obtained. Replacing β-mercaptoethanol with less volatile dithiothreitol in lysis buffer did not lead to a decrease in the yield. We compared the method proposed with miRNeasy Mini Kit (Qiagen) for isolation of microRNA from human blood plasma. MicroRNA yield was evaluated by the difference in median Ct values obtained for exogenous cel-238 and endogenous microRNA-21 cDNA amplification. For both tested microRNA, the precipitation from one-phase lysate provided better recovery with lower Ct values (Δ median Ct 4.94 for cel-238, р = 1,0E-04 and Δ median Ct 2.18 for microRNA-21, р = 9,0E-04). Thus, the method we described showed high yield and operating convenience because it can be applied without special equipment.

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Section: Resultssupporting

confidence: 92%

One-phase phenol-free method for microRNA isolation from blood plasma

et al. 2018

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“…In agreement with our results, RE has been reported to provide high-quality RNA from T lymphocytes (RIN value of 10) [27], from bacterial cells (RIN values between 8.75-9.65) [28,29,30], as well as from yeast cells (RIN value of 10) [31]. Lower RIN values of 8 and 7 were obtained for RE-based RNA extraction from human parotid tissue after snap-freezing storage with and without pre-treatment in RNAlater, respectively [32].…”

Section: Pbmc Storage Medium Rna Extraction Kit (Elution Volume; Finasupporting

confidence: 93%

Comparison of procedures for RNA-extraction from peripheral blood mononuclear cells

et al. 2020

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RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Thus far, only a limited number of methodological studies have compared sample storage and RNA extraction procedures for human cells. We compared three commercially available RNA extraction kits, i.e., (Nucli-SENS) easyMAG, RNeasy (Mini Kit) and RiboPure (RNA Purification Kit-blood). In addition, additional conditions, such as storage medium and storage temperature of human peripheral blood mononuclear cells were evaluated, i.e., 4˚C for RNAlater or -80˚C for QIAzol and for the respective cognate lysis buffers; easyMAG, RNeasy or RiboPure. RNA was extracted from aliquots that had been stored for one day (Run 1) or 83 days (Run 2). After DNase treatment, quantity and quality of RNA were assessed by means of a NanoDrop spectrophotometer, 2100 Bioanalyzer and RT-qPCR for the ACTB reference gene. We observed that high-quality RNA can be obtained using RNeasy and RiboPure, regardless of the storage medium, whereas samples stored in RNAlater resulted in the least amount of RNA extracted. In addition, RiboPure combined with storage of samples in its cognate lysis buffer yielded twice as much RNA as all other procedures. These results were supported by RT-qPCR and by the reproducibility observed for two independent extraction runs.PLOS ONE | https://doi.org/10.1371/journal.pone.

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“…Chaotropic salts such as guanidium thyocyanate are commonly used in nucleic acid purification as they disrupt membranes and denature RNases. 29,30 Solution-based extraction methods lead to higher yields of DNA, but also to a higher variability. On the other hand, column-based methods lead to slightly lower yields, but have a more consistent performance, as shown by the PT program results.…”

Section: Comparison Of Processing Methods: Nucleic Acid Extractionmentioning

confidence: 99%

A Biospecimen Proficiency Testing Program for Biobank Accreditation: Four Years of Experience

Gaignaux

Ashton

Coppola

et al. 2016

Biopreservation and Biobanking

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Biobanks produce and distribute biospecimens, ensuring their fitness for purpose and accurately qualifying them before distribution. In their efforts toward professionalization, biobanks can nowadays seek certification or accreditation. One of the requirements of these standards is regular participation in Proficiency Testing (PT) programs. An international PT program has been developed and provided to biobanks and other laboratories that perform specific tests to qualify different types of biospecimens. This PT program includes biospecimen testing schemes, as well as biospecimen processing interlaboratory exercises. This PT program supports the development of biobank quality assurance by providing the possibility to assess biobank laboratory performance and useful insights into biobank laboratory method performance characteristics and thus fulfill the demands from accreditation authorities.

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Replacing β-mercaptoethanol in RNA extractions

Cited by 27 publications

References 7 publications

One-phase phenol-free method for microRNA isolation from blood plasma

One-phase phenol-free method for microRNA isolation from blood plasma

Comparison of procedures for RNA-extraction from peripheral blood mononuclear cells

A Biospecimen Proficiency Testing Program for Biobank Accreditation: Four Years of Experience

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