Measuring microRNAs: Comparisons of microarray and quantitative PCR measurements, and of different total RNA prep methods

<p>Abstract</p> <p>Background</p> <p>Determining the expression levels of microRNAs (miRNAs) is of great interest to researchers in many areas of biology, given the significant roles these molecules play in cellular regulation. Two common methods for measuring miRNAs in a total RNA sample are microarrays and quantitative RT-PCR (qPCR). To understand the results of studies that use these two different techniques to measure miRNAs, it is important to understand how well the results of these two analysis methods correlate. Since both methods use total RNA as a starting material, it is also critical to understand how measurement of miRNAs might be affected by the particular method of total RNA preparation used.</p> <p>Results</p> <p>We measured the expression of 470 human miRNAs in nine human tissues using Agilent microarrays, and compared these results to qPCR profiles of 61 miRNAs in the same tissues. Most expressed miRNAs (53/60) correlated well (R > 0.9) between the two methods. Using spiked-in synthetic miRNAs, we further examined the two miRNAs with the lowest correlations, and found the differences cannot be attributed to differential sensitivity of the two methods. We also tested three widely-used total RNA sample prep methods using miRNA microarrays. We found that while almost all miRNA levels correspond between the three methods, there were a few miRNAs whose levels consistently differed between the different prep techniques when measured by microarray analysis. These differences were corroborated by qPCR measurements.</p> <p>Conclusion</p> <p>The correlations between Agilent miRNA microarray results and qPCR results are generally excellent, as are the correlations between different total RNA prep methods. However, there are a few miRNAs whose levels do not correlate between the microarray and qPCR measurements, or between different sample prep methods. Researchers should therefore take care when comparing results obtained using different analysis or sample preparation methods.</p

Robust interlaboratory reproducibility of a gene expression signature measurement consistent with the needs of a new generation of diagnostic tools

The increasing use of DNA microarrays in biomedical research, toxicogenomics, pharmaceutical development, and diagnostics has focused attention on the reproducibility and reliability of microarray measurements. While the reproducibility of microarray gene expression measurements has been the subject of several recent reports, there is still a need for systematic investigation into what factors most contribute to variability of measured expression levels observed among different laboratories and different experimenters.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

lincRNAs act in the circuitry controlling pluripotency and differentiation

Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ES cell regulators. Notably, lincRNAs primarily affect gene expression in trans. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ES cells and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ES cell state.Broad InstituteHarvard UniversityNational Human Genome Research Institute (U.S.)Merkin Family Foundation for Stem Cell Researc

Direct and sensitive miRNA profiling from low-input total RNA

We have developed a sensitive, accurate, and multiplexed microRNA (miRNA) profiling assay that is based on a highly efficient labeling method and novel microarray probe design. The probes provide both sequence and size discrimination, yielding in most cases highly specific detection of closely related mature miRNAs. Using a simple, single-vial experimental protocol, 120 ng of total RNA is directly labeled using Cy3 or Cy5, without fractionation or amplification, to produce precise and accurate measurements that span a linear dynamic range from 0.2 amol to 2 fmol of input miRNA. The results can provide quantitative estimates of the miRNA content for the tissues studied. The assay is also suitable for use with formalin-fixed paraffin-embedded clinical samples. Our method allows rapid design and validation of probes for simultaneous quantitative measurements of all human miRNA sequences in the public databases and to new miRNA sequences as they are reported

Use of complex oligonucleotide libraries for concurrent high-resolution fluorescence imaging of both DNA and RNA in various sample types

Fluorescence in situ Hybridization (FISH) is a powerful technique for determining the localization specific nucleic acid sequences within individual cells. Previously, the use of FISH has often been dependent upon access to cloned template DNA for the generation of probes, which can be difficult if clones for specific regions are unavailable, or if the genomic region of interest contains repetitive and/or other problematic sequences. We have developed the ability to chemically synthesize DNA in massively parallel reactions, which we have used to produce libraries of oligonucleotides up to 200 bases in length that can be utilized for the generation of FISH probes. The sequences of the oligonucleotides in these libraries are selected in silico using empirically determined criteria so as to avoid repetitive elements or regions homologous to other non-targeted loci. We have found that these oligonucleotide library-derived FISH probes can detect human genomic regions as small as 1.8 kb and as large as whole chromosomes in both metaphase and interphase cells, using the same simple assay protocol. Because of the inherent flexibility in our probe design methods, we can readily visualize regions rich in repeats and/or GC content. We have also used these oligonucleotide library-derived FISH probes to detect the localization of a variety of both coding and non-coding RNAs in fixed tissue culture cells and formalin-fixed paraffin-embedded tissue sections, using both conventional fluorescence and structured illumination microscopy. Simultaneous hybridization of FISH probes labeled with different fluorophores enables visualization of multiple sequences at once. Using probes designed specifically to transcribed vs. non-transcribed regions has enabled the simultaneously detect DNA and RNA from the same locus, or from two different loci, in the same FISH assay. The ability to generate high performance FISH probes using chemically synthesized oligo libraries that can simultaneously detect DNA and RNA yields a valuable tool for studies of how localization of specific nucleic acids impacts biological function

A Prader-Willi locus lncRNA cloud modulates diurnal genes and energy expenditure.

Prader-Willi syndrome (PWS), a genetic disorder of obesity, intellectual disability and sleep abnormalities, is caused by loss of non-coding RNAs on paternal chromosome 15q11-q13. The imprinted minimal PWS locus encompasses a long non-coding RNA (lncRNA) transcript processed into multiple SNORD116 small nucleolar RNAs and the spliced exons of the host gene, 116HG. However, both the molecular function and the disease relevance of the spliced lncRNA 116HG are unknown. Here, we show that 116HG forms a subnuclear RNA cloud that co-purifies with the transcriptional activator RBBP5 and active metabolic genes, remains tethered to the site of its transcription and increases in size in post-natal neurons and during sleep. Snord116del mice lacking 116HG exhibited increased energy expenditure corresponding to the dysregulation of diurnally expressed Mtor and circadian genes Clock, Cry1 and Per2. These combined genomic and metabolic analyses demonstrate that 116HG regulates the diurnal energy expenditure of the brain. These novel molecular insights into the energy imbalance in PWS should lead to improved therapies and understanding of lncRNA roles in complex neurodevelopmental and metabolic disorders