Short Structured RNAs with Low GC Content Are Selectively Lost during Extraction from a Small Number of Cells

Kim, Young‐Kook; Yeo, Jinah; Kim, Boseon; Ha, Minju; Kim, V. Narry

doi:10.1016/j.molcel.2012.05.036

Cited by 196 publications

(153 citation statements)

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“…It should be noted that RNA extraction from a small number of cells by organic extraction method could be selectively inefficient for small structured RNAs, such as tRNAs. 29 Thus, total RNA for FL-PCR quantification should be extracted from high amount of cells or identical number of cells when comparing mature tRNA levels in different samples.…”

Section: Resultsmentioning

confidence: 99%

Four-leaf clover qRT-PCR: A convenient method for selective quantification of mature tRNA

et al. 2015

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Transfer RNAs (tRNAs) play a central role in translation and also recently appear to have a variety of other functions in biological processes beyond translation. Here we report the development of Four-Leaf clover qRT-PCR (FL-PCR), a convenient PCR-based method, which can specifically quantify individual mature tRNA species. In FL-PCR, T4 RNA ligase 2 specifically ligates a stem-loop adapter to mature tRNAs but not to precursor tRNAs or tRNA fragments. Subsequent TaqMan qRT-PCR amplifies only unmodified regions of the tRNA-adapter ligation products; therefore, FL-PCR quantification is not influenced by tRNA post-transcriptional modifications. FL-PCR has broad applicability for the quantification of various tRNAs in different cell types, and thus provides a much-needed simple method for analyzing tRNA abundance and heterogeneity.

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Section: Resultsmentioning

confidence: 99%

Four-leaf clover qRT-PCR: A convenient method for selective quantification of mature tRNA

et al. 2015

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“…Tissue samples were removed from storage and placed in 1 mL TRI Reagent ® solution supplemented with the addition of 10 m m MgCl 2 to aid recovery of microRNAs for future analysis (Kim et al ., 2012). Samples were then completely homogenized (15mins for spleen samples, 30mins for muscle samples) using a bead mill (Retsch Technology GmbH, Haan, Germany).…”

Section: Methodsmentioning

confidence: 99%

Changes in the expression of splicing factor transcripts and variations in alternative splicing are associated with lifespan in mice and humans

Lee¹,

Pilling

Emond³

et al. 2016

Aging Cell

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SummaryDysregulation of splicing factor expression and altered alternative splicing are associated with aging in humans and other species, and also with replicative senescence in cultured cells. Here, we assess whether expression changes of key splicing regulator genes and consequent effects on alternative splicing are also associated with strain longevity in old and young mice, across 6 different mouse strains with varying lifespan (A/J, NOD.B10Sn‐H2b/J, PWD.Phj, 129S1/SvlmJ, C57BL/6J and WSB/EiJ). Splicing factor expression and changes to alternative splicing were associated with strain lifespan in spleen and to a lesser extent in muscle. These changes mainly involved hnRNP splicing inhibitor transcripts with most changes more marked in spleens of young animals from long‐lived strains. Changes in spleen isoform expression were suggestive of reduced cellular senescence and retained cellular proliferative capacity in long‐lived strains. Changes in muscle isoform expression were consistent with reduced pro‐inflammatory signalling in longer‐lived strains. Two splicing regulators, HNRNPA1 and HNRNPA2B1, were also associated with parental longevity in humans, in the InCHIANTI aging study. Splicing factors may represent a driver, mediator or early marker of lifespan in mouse, as expression differences were present in the young animals of long‐lived strains. Changes to alternative splicing patterns of key senescence genes in spleen and key remodelling genes in muscle suggest that correct regulation of alternative splicing may enhance lifespan in mice. Expression of some splicing factors in humans was also associated with parental longevity, suggesting that splicing regulation may also influence lifespan in humans.

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“…Indeed, the effect of extraction technique on miRNA expression detection has been previously reported by others. 33 The identification of deregulated miRNAs common to both tumor sets, despite the different methodologies employed, adds weight to the evidence that they play a role in thyroid cancer.…”

Section: Modern Pathology (2015) 28 748-757mentioning

confidence: 99%

MicroRNA deep-sequencing reveals master regulators of follicular and papillary thyroid tumors

et al. 2015

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MicroRNA deregulation could be a crucial event in thyroid carcinogenesis. However, current knowledge is based on studies that have used inherently biased methods. Thus, we aimed to define in an unbiased way a list of deregulated microRNAs in well-differentiated thyroid cancer in order to identify diagnostic and prognostic markers. We performed a microRNA deep-sequencing study using the largest well-differentiated thyroid tumor collection reported to date, comprising 127 molecularly characterized tumors with follicular or papillary patterns of growth and available clinical follow-up data, and 17 normal tissue samples. Furthermore, we integrated microRNA and gene expression data for the same tumors to propose targets for the novel molecules identified. Two main microRNA expression profiles were identified: one common for follicular-pattern tumors, and a second for papillary tumors. Follicular tumors showed a notable overexpression of several members of miR-515 family, and downregulation of the novel microRNA miR-1247. Among papillary tumors, top upregulated microRNAs were miR-146b and the miR-221~222 cluster, while miR-1179 was downregulated. BRAF-positive samples displayed extreme downregulation of miR-7 and -204. The identification of the predicted targets for the novel molecules gave insights into the proliferative potential of the transformed follicular cell. Finally, by integrating clinical follow-up information with microRNA expression, we propose a prediction model for disease relapse based on expression of two miRNAs (miR-192 and let-7a) and several other clinicopathological features. This comprehensive study complements the existing knowledge about deregulated microRNAs in the development of well-differentiated thyroid cancer and identifies novel markers associated with recurrence-free survival.

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Short Structured RNAs with Low GC Content Are Selectively Lost during Extraction from a Small Number of Cells

Cited by 196 publications

References 1 publication

Four-leaf clover qRT-PCR: A convenient method for selective quantification of mature tRNA

Four-leaf clover qRT-PCR: A convenient method for selective quantification of mature tRNA

Changes in the expression of splicing factor transcripts and variations in alternative splicing are associated with lifespan in mice and humans

MicroRNA deep-sequencing reveals master regulators of follicular and papillary thyroid tumors

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