Comparison of different extraction techniques to profile microRNAs from human sera and peripheral blood mononuclear cells

Monleau, Marjorie; Bonnel, Sophie; Gostan, Thierry; Blanchard, Dominique; Courgnaud, Valérie; Lecellier, Charles-Henri

doi:10.1186/1471-2164-15-395

Cited by 61 publications

(43 citation statements)

References 29 publications

(33 reference statements)

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“…This issue has already been postulated by Monleau et al . Since we were able to gain the best results with the method that was optimized for isolation of bulk tRNA, we speculate that the mechanism of tRNA‐derived fragments loss during the isolation procedure is related to specific characteristics of tRNA molecules, including sequence, structure or high molecular interaction partners.…”

Section: Discussionmentioning

confidence: 99%

The widespread occurrence of tRNA‐derived fragments in Saccharomyces cerevisiae

et al. 2016

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Short RNA s derived from the cleavage of tRNA molecules are observed in most organisms. Their occurrence seems to be induced by stress conditions, but still little is known about their biogenesis and functions. We find that the recovery of tRNA fragments depends on the RNA isolation method. Using an optimized RNA extraction protocol and northern blot hybridization technique, we show that the tRNA ‐derived fragments in yeast are widespread in 12 different growth conditions. We did not observe significant stress‐dependent changes in the amounts of tRNA fragments pool. Instead, we show the differential processing of almost all individual tRNA s. We also provide evidence that 3′‐part‐derived tRNA fragments are as abundant as the 5′‐ one in Saccharomyces cerevisiae . The resulting set of S. cerevisiae tRNA fragments provides a robust basis for further experimental studies on biological functions of tRF s.

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Section: Discussionmentioning

confidence: 99%

The widespread occurrence of tRNA‐derived fragments in Saccharomyces cerevisiae

et al. 2016

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“…Despite the miRNeasy kit presents the highest theoretical concentration factor, our data showed that the NucleoSpin ® miRNA plasma kit provides higher endogenous miRNA concentrations and performs as well as the others, when reproducibility is assessed. Using high‐throughput PCR arrays, the NucleoSpin ® miRNA plasma kit has been recently described as more efficient than other kits (miRNeasy and Norgen) to isolate serum miRNAs (Monleau et al., 2014) and as presenting the least extraction bias (Tan et al., 2015) in agreement with our individual PCR assays. We provided additional data showing its higher performance on both serum and plasma miRNA isolation using individual absolute RT‐qPCR.…”

Section: Discussionmentioning

confidence: 99%

Towards a new standardized method for circulating miRNAs profiling in clinical studies: Interest of the exogenous normalization to improve miRNA signature accuracy

et al. 2016

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Circulating miRNAs are promising biomarkers in oncology but have not yet been implemented in the clinic given the lack of concordance across studies. In order to increase the cross‐studies reliability, we attempted to reduce and to control the circulating miRNA expression variability between patients. First, to maximize profiling signals and to reduce miRNA expression variability, three isolation kits were compared and the NucleoSpin® kit provided higher miRNA concentrations than the other widely used kits. Second, to control inter‐sample variability during the profiling step, the exogenous miRNAs normalization method commonly used for RT‐qPCR validation step was adapted to microarray experiments. Importantly, exogenous miRNAs presented two‐fold lower inter‐sample variability than the widely used endogenous miR‐16‐5p reflecting that the latter is subject to both biological and technical variability. Although Caenorhabditis elegans miRNAs isolation yields were heterogeneous, they correlated to each other and to their geometrical mean across samples. The normalization based on the geometrical mean of three exogenous miRNAs increased the correlation up‐to 0.97 between the microarrays and individual RT‐qPCR steps of circulating miRNAs expression. Overall, this new strategy open new avenue to identify reliable circulating miRNA signatures for translation into clinical practice.

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“…Several studies have tackled this point, focusing on methods for miRNA extraction (Monleau et al 2014;Li and Kowdley 2012;Podolska et al 2011); however, similar considerations could also be true in analyzing sdRNAs.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of methods for the detection of low-abundant snoRNA-derived small RNAs in Saccharomyces cerevisiae

Walkowiak

Mleczko

Bąkowska-Żywicka

2016

bta

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In recent years, there are a growing number of studies demonstrating the existence of small RNAs derived from snoRNAs (sdRNAs) in multiple eukaryotic organisms. Such RNAs have been initially observed in high throughput sequencing studies and assumed to be processed by miRNA machinery. Recently, we have identified sdRNAs that are associated with ribosomes in yeast Saccharomyces cerevisiae. Although sdRNAs were detectable in sequencing data, their low abundance hampered their detection by other methods. Here, we present the results of our survey for optimized experimental method for sdRNA detection. We have compared two extraction procedures of total RNA from S. cerevisiae : MasterPure TM kit and Trizol with two methods resulting in enrichment in small RNA fraction and MasterPure TM with selective isopropanol precipitation and bulk tRNA isolation methods. Also the sensitivity of three methods for sdRNA detection was verified: a northern blot using standard or LNA probes and stem-loop reverse transcription followed by PCR (SL-RT-PCR). Our results reveal that Trizol isolation method combined with SL-RT-PCR is the most effective in the detection of low-abundant sdRNAs.

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Comparison of different extraction techniques to profile microRNAs from human sera and peripheral blood mononuclear cells

Cited by 61 publications

References 29 publications

The widespread occurrence of tRNA‐derived fragments in Saccharomyces cerevisiae

The widespread occurrence of tRNA‐derived fragments in Saccharomyces cerevisiae

Towards a new standardized method for circulating miRNAs profiling in clinical studies: Interest of the exogenous normalization to improve miRNA signature accuracy

Evaluation of methods for the detection of low-abundant snoRNA-derived small RNAs in Saccharomyces cerevisiae

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