Project description:The short length of miRNAs results in a high dynamic range of melting temperatures and therefore 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 developed a new method based not only to the hybridization process that presents the limits before described, but integrating the hybridization to an enzymatic reaction. Moreover we introduced spike-in in the hybridization-enzymatic reaction allowing the quantification of miRNAs respect to them, canceling biases related to sequence, labeling, or hybridization. An alternative method for the absolute miRNA quantization was recently proposed by Bissels (Absolute quantification of microRNAs by using a universal reference. RNA). It was based on the Absolute quantification of microRNAs by using a universal reference consisting of 954 synthetic human, mouse, rat, and viral miRNAs, with each individual oligoribonucleotide present in equimolar concentrations with tested miRNAs. Thereby, any single miRNA detected on a microarray can be quantified by directly comparing its signal intensity with the one obtained by the same miRNA sequence present in the universal reference adjusting for biases related to sequence, labeling, hybridization, or signal detection. Our method allowed the detection of a comparable concentration of miRNA (10-18 moles to 10-14 moles in a linear range) (see Figure), but allows controlling the hybridization quality and reproducibility basing on the results of the interpolation of the spike-in dependent curve. Moreover, our method does not influenced by phenomena imputable to different labeling process due to different sequences because labeling was due only to the incorporation of biotin-d(A) if the hybridized miRNA acted as primer for the klenow enzyme. This method allowed the discussion of miRNA genes expression in 14 different tissues relating it with tissue anatomical proximity and functional similarity. 2K microarray was hybridized with small RNAs from 14 different tissues (Atrium Sinister; Skin; Liver; W_B_A: White Blood Cells from Arteriosum blood; W_B_V: White Blood Cells from Venosum blood; Lymph Node; Tongue; Spleen; Skeletal Muscle; Lung; Kidney; Stomach; Adipose Tissue; Ventricle Sinister). Each experiment was replicated to have 28 experiments and miRNA gene expression was correlated with mRNA gene expression in the same samples.

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. 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).

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:The short length of miRNAs results in a high dynamic range of melting temperatures and therefore 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 developed a new method based not only to the hybridization process that presents the limits before described, but integrating the hybridization to an enzymatic reaction. Moreover we introduced spike-in in the hybridization-enzymatic reaction allowing the quantification of miRNAs respect to them, canceling biases related to sequence, labeling, or hybridization. An alternative method for the absolute miRNA quantization was recently proposed by Bissels (Absolute quantification of microRNAs by using a universal reference. RNA). It was based on the Absolute quantification of microRNAs by using a universal reference consisting of 954 synthetic human, mouse, rat, and viral miRNAs, with each individual oligoribonucleotide present in equimolar concentrations with tested miRNAs. Thereby, any single miRNA detected on a microarray can be quantified by directly comparing its signal intensity with the one obtained by the same miRNA sequence present in the universal reference adjusting for biases related to sequence, labeling, hybridization, or signal detection. Our method allowed the detection of a comparable concentration of miRNA (10-18 moles to 10-14 moles in a linear range) (see Figure), but allows controlling the hybridization quality and reproducibility basing on the results of the interpolation of the spike-in dependent curve. Moreover, our method does not influenced by phenomena imputable to different labeling process due to different sequences because labeling was due only to the incorporation of biotin-d(A) if the hybridized miRNA acted as primer for the klenow enzyme. This method allowed the discussion of miRNA genes expression in 14 different tissues relating it with tissue anatomical proximity and functional similarity.

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MiRNA-cardiocirculatory

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