Replacing cRNA targets with cDNA reduces microarray cross-hybridization

Eklund, Aron C.; Turner, Leah; Chen, Pengchin; Jensen, Roderick V.; deFeo, Gianfranco; Kopf-Sill, Anne; Szállási, Zoltán

doi:10.1038/nbt0906-1071

Cited by 60 publications

(57 citation statements)

References 19 publications

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“…cRNA targets have been shown to produce lower perfect match/mismatch discrimination scored than cDNA targets and therefore many elements are not called present with cRNA targets despite having signals significantly above the background. Several publications address this observation including the publications by Barker et al 25 and Eklund et al 26 More information is being gained from less input RNA using this new method. This is supported by the higher percent present calls and also the selection of statistically significant genes that discriminate the two biological subtypes of DLBCL (ABC and GCB).…”

Section: Discussionmentioning

confidence: 99%

A Novel Method of Amplification of FFPET-Derived RNA Enables Accurate Disease Classification with Microarrays

Williams

Johnson

et al. 2010

The Journal of Molecular Diagnostics

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Section: Discussionmentioning

confidence: 99%

A Novel Method of Amplification of FFPET-Derived RNA Enables Accurate Disease Classification with Microarrays

Williams

Johnson

et al. 2010

The Journal of Molecular Diagnostics

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“…Expression levels of 220 human miRNAs were measured by custom quantitative PCR assays, as described previously (15,16). Transcript expression levels were detected by microarray, as described previously (17,18). Seven mice were analyzed for each treatment or control group.…”

Section: Methodsmentioning

confidence: 99%

DLK1-DIO3 Genomic Imprinted MicroRNA Cluster at 14q32.2 Defines a Stemlike Subtype of Hepatocellular Carcinoma Associated with Poor Survival

Luk

Burchard

Zhang

et al. 2011

Journal of Biological Chemistry

144

123

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Hepatocellular carcinoma (HCC) is a heterogeneous and highly aggressive malignancy, for which there are no effective cures. Identification of a malignant stemlike subtype of HCC may offer patients with a dismal prognosis a potential targeted therapy using c-MET and Wnt pathway inhibitors. MicroRNAs (miRNAs) show promise as diagnostic and prognostic tools for cancer detection and stratification. Using a TRE-c-Met-driven transgenic HCC mouse model, we identified a cluster of 23 miRNAs that is encoded within the Dlk1-Gtl2 imprinted region on chromosome 12qF1 overexpressed in all of the isolated liver tumors. Interestingly, this region is conserved among mammalian species and maps to the human DLK1-DIO3 region on chromosome 14q32.2. We thus examined the expression of the DLK1-DIO3 miRNA cluster in a cohort of 97 hepatitis B virus-associated HCC patients and identified a subgroup (n ‫؍‬ 18) of patients showing strong coordinate overexpression of miRNAs in this cluster but not in other cancer types (breast, lung, kidney, stomach, and colon) that were tested. Expression levels of imprinted gene transcripts from neighboring loci in this 14q32.2 region and from a subset of other imprinted sites were concomitantly elevated in human HCC. Interestingly, overexpression of the DLK1-DIO3 miRNA cluster was positively correlated with HCC stem cell markers (CD133, CD90, EpCAM, Nestin) and associated with a high level of serum ␣-fetoprotein, a conventional biomarker for liver cancer, and poor survival rate in HCC patients. In conclusion, our findings suggest that coordinate up-regulation of the DLK1-DIO3 miRNA cluster at 14q32.2 may define a novel molecular (stem cell-like) subtype of HCC associated with poor prognosis.

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“…This limits the ability of the method to measure the relative concentration of distinct RNAs within the same sample and requires the blot to be stripped and reprobed. Microarrays are ideal for the comparison of relative RNA concentrations within a sample, but microarrays are costly and tend, due to hybridization kinetics, to suffer from a relatively low dynamic range (Zhang et al 2005;Eklund et al 2006;MAQC Consortium et al 2006). Methods based on quantitative PCR are likely to be very sensitive but require multiple enzymatic steps and multiple pairs of oligonucleotides to achieve results.…”

Section: Introductionmentioning

confidence: 99%

Characterizing multiple exogenous and endogenous small RNA populations in parallel with subfemtomolar sensitivity using a streptavidin gel-shift assay

Ebhardt¹,

Unrau²

2009

RNA

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Here we present a simple and inexpensive gel-shift assay for the detection and quantification of small RNAs. The assay is at least 5-10 times more sensitive than a conventional Northern, and is highly scalable. Total RNA is first size purified to enrich the desired size range, phosphatase treated, and then radiolabeled to high specific activity using polynucleotide kinase. The resulting RNA stock is then hybridized to an excess of biotinylated DNA probe oligonucleotide, prior to mixing with streptavidin and loading on a native gel. The amount of supershifted material was proportional to the amount of labeled target RNA in the sample. We applied this method to verify sequencing data originally obtained from a four-point comparison study on the effect of endogenous expression of HC-Pro on Y-satellite/cucumber mosaic virus infection in tobacco plants. The results of the streptavidin gel-shift assay were consistent with the concentrations of small RNA infected plants inferred by our original cloning data, and rapidly provided information about the relative concentration of a number of viral and endogenous small RNAs. Further straightforward improvements to this simple methodology might be expected to improve the methods sensitivity by as much as another 10-fold.

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Replacing cRNA targets with cDNA reduces microarray cross-hybridization

Cited by 60 publications

References 19 publications

A Novel Method of Amplification of FFPET-Derived RNA Enables Accurate Disease Classification with Microarrays

A Novel Method of Amplification of FFPET-Derived RNA Enables Accurate Disease Classification with Microarrays

DLK1-DIO3 Genomic Imprinted MicroRNA Cluster at 14q32.2 Defines a Stemlike Subtype of Hepatocellular Carcinoma Associated with Poor Survival

Characterizing multiple exogenous and endogenous small RNA populations in parallel with subfemtomolar sensitivity using a streptavidin gel-shift assay

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