Absolute mRNA concentrations from sequence-specific calibration of oligonucleotide arrays

Hekstra, Doeke R.; Taussig, Alexander R.; Magnasco, Marcelo O.; Naef, Félix

doi:10.1093/nar/gkg283

Cited by 151 publications

(165 citation statements)

References 9 publications

Supporting

Mentioning

159

Contrasting

Order By: Relevance

“…Therefore, although the different available microarray platforms for mRNA expression profiling have been extensively evaluated (Canales et al 2006), there is still no gold standard for quantifying mRNA transcript concentrations by microarrays. The approaches developed so far encompass mathematical modeling (Hekstra et al 2003;Frigessi et al 2005), calibrated reference samples (Dudley et al 2002), exogenous RNA controls (Carter et al 2005b), and combinations of mathematical modeling and spike measurements (Zhao et al 2007). The TransCount method (Frigessi et al 2005) is based on Bayesian statistical modeling and utilizes covariants of the microarray experiment to calculate the concentration from the signal intensity of each probe.…”

Section: Discussionmentioning

confidence: 99%

Absolute quantification of microRNAs by using a universal reference

Bissels¹,

Wild²,

Tomiuk³

et al. 2009

RNA

178

132

View full text Add to dashboard Cite

MicroRNAs (miRNAs) are a species of small RNAs ;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 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 the parallel hybridization of the sample of interest labeled with Cy5 and a universal reference of 954 synthetic miRNAs in equimolar concentrations that are labeled with Cy3 on a microarray slide containing probes for all human, mouse, rat, and viral miRNAs (miRBase 12.0). Each single miRNA is quantified with respect to the universal reference canceling biases related to sequence, labeling, or hybridization. We demonstrate the accuracy of the method by various spike-in experiments. Furthermore, we quantified miRNA copy numbers in liver samples and CD34(+)/ CD133(À) hematopoietic progenitor cells.

show abstract

Section: Discussionmentioning

confidence: 99%

Absolute quantification of microRNAs by using a universal reference

Bissels¹,

Wild²,

Tomiuk³

et al. 2009

RNA

178

132

View full text Add to dashboard Cite

show abstract

“…55,56 For example, Corn and co-workers fit thermodynamic data for probes on planar Au to a Langmuir isotherm and report similar K d values (55 nM for an 18-mer probe/target duplex in a 300 mM NaCl, 100 mM urea PBS buffer). 57 Peterson et al found that perfectly matched duplex formation on an Au surface could be fit to a Langmuir isotherm (with K d =17 nM for a 25-mer duplex in 1 M NaCl buffer), while mismatched duplexes were better modeled by the Sips isotherm.…”

Section: Nanowire Mb Assay Sensitivitymentioning

confidence: 94%

Coupling Molecular Beacons to Barcoded Metal Nanowires for Multiplexed, Sealed Chamber DNA Bioassays

et al. 2006

View full text Add to dashboard Cite

We have combined molecular beacon (MB) probes with barcoded metal nanowires to enable nowash, sealed chamber, multiplexed detection of nucleic acids. Probe design and experimental parameters important in nanowire-based MB assays are discussed. Loop regions of 24 bases and 5 base pair stem regions in the beacon probes gave optimal performance. Our results suggest that thermodynamic predictions for secondary structure stability of solution-phase MB can guide probe design for nanowire-based assays. Dengue virus-specific probes with predicted solution-phase ΔG of folding in 500 mM buffered NaCl of approximately −4 kcal/mol performed better than those with ΔG > −2 or < −6 kcal/mol. Buffered 300-500 mM NaCl was selected after comparison of several buffers previously reported for similar types of assays, and 200-500 mM NaCl was found to be the optimal ionic strength for the hybridization temperatures (25 and 50 °C) and probe designs used here. Target binding to the surface as a function of solution concentration fit a Sips isotherm with K d = 1.7 ± 0.3 nM. The detection limit was ∼100 pM, limited by incomplete quenching. Single base mismatches could be discriminated from fully complementary targets. Oligonucleotide target sequences specific for human immunodeficiency, hepatitis C, and severe acute respiratory viruses were assayed simultaneously in a no-wash, sealed chamber, multiplexed experiment in which each of three probe sequences was attached to a different pattern of encoded nanowires. Finally, we demonstrated that probe-coated nanowires retain their selectivity and sensitivity in a triplexed assay after storage for over 3 months.

show abstract

“…Whereas the former results were based on the outcome of hybridization experiments only, thermodynamics has been taken into account in several other studies [6][7][8][9][10][11][12][13][14][15][16][17][18][19] . Indeed, hybridization of nucleotide molecules in solution is a well understood biophysical process driven by thermodynamics 20 .…”

Section: Introductionmentioning

confidence: 99%

Thermodynamic Behavior of Short Oligonucleotides in Microarray Hybridizations Can Be Described Using Gibbs Free Energy in a Nearest-Neighbor Model

et al. 2007

View full text Add to dashboard Cite

While designing oligonucleotide-based microarrays, cross-hybridization between surface-bound oligos and non-intended labeled targets is probably the most difficult parameter to predict. Although literature describes rules-of-thumb concerning oligo length, overall similarity, and continuous stretches, the final behavior is difficult to predict. The aim of this study was to investigate the effect of well-defined mismatches on hybridization specificity using CodeLink Activated Slides, and to study quantitatively the relation between hybridization intensity and Gibbs free energy (∆G), taking the mismatches into account. Our data clearly showed a correlation between the hybridization intensity and ∆G of the oligos over three orders of magnitude for the hybridization intensity, which could be described by the Langmuir model. As ∆G was calculated according to the nearest-neighbor model, using values related to DNA hybridizations in solution, this study clearly shows that target-probe hybridizations on microarrays with a three-dimensional coating are in quantitative agreement with the corresponding reaction in solution. These results can be interesting for some practical applications. The correlation between intensity and ∆G can be used in quality control of microarray hybridizations by designing probes and corresponding RNA spikes with a range of ∆G values. Furthermore, this correlation might be of use to fine-tune oligonucleotide design algorithms in a way to improve the prediction of the influence of mismatching targets on microarray hybridizations. KEYWORDSMicroarray oligonucleotide design, nearest-neighbor model, Langmuir model, microarray quality control 3

show abstract

Absolute mRNA concentrations from sequence-specific calibration of oligonucleotide arrays

Cited by 151 publications

References 9 publications

Absolute quantification of microRNAs by using a universal reference

Absolute quantification of microRNAs by using a universal reference

Coupling Molecular Beacons to Barcoded Metal Nanowires for Multiplexed, Sealed Chamber DNA Bioassays

Thermodynamic Behavior of Short Oligonucleotides in Microarray Hybridizations Can Be Described Using Gibbs Free Energy in a Nearest-Neighbor Model

Contact Info

Product

Resources

About