Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Microarray probe design

ABSTRACT: MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells. Different probe designs were investigated with respect to sensitivity and selectivity of microarray hybridization. An array with 11 different variants of oligonucleotides for miR-122a and miR-16 was produced. 5 µg of respective total RNA was fluorescently labelled by 3’ ligation. Total RNA was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool. The synthetic miRNA pool consisted of 5 fmol of each of 816 non redundant miRNAs sequences and miRControl 3 sequences. Local background was subtracted from the signal to obtain the net signal intensity and the mean of the net signal intensities of 4 corresponding spots representing the same miRNA was computed for those spots only which were unflagged (empty spots, poor spots, negative spots) and for which the fluorescent intensity of the miRNAs derived from the samples of interest was two-fold the mean background value (2bkg dataset).

ORGANISM(S): synthetic construct

SUBMITTER: Ute Bissels

PROVIDER: E-GEOD-15832 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Absolute quantification of microRNAs by using a universal reference.

Bissels Ute U Wild Stefan S Tomiuk Stefan S Holste Angela A Hafner Markus M Tuschl Thomas T Bosio Andreas A

RNA (New York, N.Y.) 20091027 12

MicroRNAs (miRNAs) are a species of small RNAs approximately 21-23-nucleotides long that have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous PCR-, sequencing-, or hybridization-based methods have been established to identify and quantify miRNAs. Their short length results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While mi ...[more]

PMID: 19861428

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Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells. The RNA extracted from 7 x 10^ 5 to 1 x 10^ 6 CD34(+)/CD133(-) cells of three different donors was analyzed. 1 µg of respective total RNA was mixed with 2.5 fmol of each of 18 RNA oligonucleotides reverse complement to miRControl 3 probes and subsequently fluorescently labelled by 3’ ligation. Total RNA mix was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool. The synthetic miRNA pool consisted of 2.5 fmol of each of 954 non redundant miRNAs sequences and miRControl 3 sequences. The array data was normalized by calculating the median of the miRControl 3 present in the CD34(+)/CD133(-) and UR sample. The miRNA amount was calculated with respect to the corresponding miRNA in the UR.

Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells. We analyzed to which extend the universal reference can be used as a tool for the relative quantification of miRNAs across multiple experiments. We compared the results of direct hybridizations i.e. sample vs. sample to those of indirect hybridizations i.e. sample vs. UR. For the direct hybridizations, we hybridized 5Âµg liver total RNA vs 5 Âµg brain total RNA (n = 3) and for the indirect hybridization 5 Âµg liver or brain total RNA vs UR (5 fmol/miRNA) (n = 3). We calculated the so-called re-ratios for the UR experiments by dividing the signal ratios of the liver vs. UR array by the respective brain vs. UR array gaining a liver vs. brain re-ratio. Each RNA sample was mixed with 5 fmol of each of 18 RNA oligonucleotides reverse complement to miRControl 3 probes and subsequently fluorescently labelled. The RNA mix was hybridized in a dual colour approach to microarrays. The mean ratios of all probes were normalized to the median of the ratios detected for the spiked 18 synthetic RNA oligonucleotides reverse complement to miRControl 3 probes.

Project description:Modification of microRNA sequences by the 3' addition of nucleotides to generate so-called “isomiRs” adds to the complexity of miRNA function, with recent reports showing that 3' modifications can influence miRNA stability and efficiency of target repression. Here we show that the 3' modification of miRNAs is a physiological and common post-transcriptional event that shows selectivity for specific miRNAs and is observed across species ranging from C. elegans to human. The modifications result predominantly from adenylation and uridylation, and are seen across tissue types, disease states, and developmental stages. To quantitatively profile 3' nucleotide additions, we developed and validated a novel assay based on NanoString Technologies' nCounter platform. For certain miRNAs, the frequency of modification was altered by processes such as cell differentiation, indicating that 3' modification is a biologically regulated process. To investigate the mechanism of 3' nucleotide additions, we used RNA interference to screen a panel of eight candidate miRNA nucleotidyl transferases for 3' miRNA modification activity in human cells. Multiple enzymes, including PAPD1, PAPD4, PAPD5, ZCCHC6, ZCCHC11, and TUT1, were found to govern 3' nucleotide addition to miRNAs in a miRNA-specific manner. Three of these enzymes–PAPD1, ZCCHC6 and TUT1–have not previously been known to modify miRNAs. Collectively, our results indicate that 3' modification observed in next generation small RNA sequencing data is a biologically relevant process, and identify enzymatic mechanisms that may lead to new approaches for modulating miRNA activity in vivo. The matrix presents the counts from when the two pools of synthetic oligos were profiled in the each of the three bridge pools. We validated the specificity of the platform for the miRNAs of interest by assaying two pools of synthetic RNA oligonucleotides representing canonical or 3' variant versions of 14 miRNAs. Each bridge pool (i.e., canonical, variant 1 or variant 2 sequences) was used to assay the two mixtures of synthetic miRNA oligonucleotides. A single replicate of each pool was profiled in each assay.

Project description:Modification of microRNA sequences by the 3' addition of nucleotides to generate so-called M-bM-^@M-^\isomiRsM-bM-^@M-^] adds to the complexity of miRNA function, with recent reports showing that 3' modifications can influence miRNA stability and efficiency of target repression. Here we show that the 3' modification of miRNAs is a physiological and common post-transcriptional event that shows selectivity for specific miRNAs and is observed across species ranging from C. elegans to human. The modifications result predominantly from adenylation and uridylation, and are seen across tissue types, disease states, and developmental stages. To quantitatively profile 3' nucleotide additions, we developed and validated a novel assay based on NanoString Technologies' nCounter platform. For certain miRNAs, the frequency of modification was altered by processes such as cell differentiation, indicating that 3' modification is a biologically regulated process. To investigate the mechanism of 3' nucleotide additions, we used RNA interference to screen a panel of eight candidate miRNA nucleotidyl transferases for 3' miRNA modification activity in human cells. Multiple enzymes, including PAPD1, PAPD4, PAPD5, ZCCHC6, ZCCHC11, and TUT1, were found to govern 3' nucleotide addition to miRNAs in a miRNA-specific manner. Three of these enzymesM-bM-^@M-^SPAPD1, ZCCHC6 and TUT1M-bM-^@M-^Shave not previously been known to modify miRNAs. Collectively, our results indicate that 3' modification observed in next generation small RNA sequencing data is a biologically relevant process, and identify enzymatic mechanisms that may lead to new approaches for modulating miRNA activity in vivo. We validated the specificity of the platform by assaying three pools of synthetic RNA oligonucleotides representing canonical, variant 1, and variant 2 versions of 5 miRNAs (miR-15a, miR-15b, miR-125a-5p, miR-143 and miR-221).Each bridge pool (i.e., specific to canonical, variant 1 or variant 2) was used to assay the three mixtures of synthetic miRNA oligonucleotides. In order to assess accuracy, we also assayed mixtures of 5 synthetic miRNAs containing 60% canonical miRNA, 30% variant 1, and 10% variant 2. Triplicate technical replicates of each sample were performed.

Dataset Information

Microarray probe design

Publications

Absolute quantification of microRNAs by using a universal reference.

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