Ultrasensitive RNA profiling: Counting single molecules on microarrays

Mir, Kalim U.

doi:10.1101/gr.5825506

Cited by 18 publications

(17 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It should be noted however, that it would not be necessary to achieve reaction completion at each cycle and dephasing would not be a problem if analysis was at the single molecule level. Single molecule detection would enable the asynchronous template-directed extension of each primer molecule within the spot to be monitored independently irrespective of its extent of extension; this would be followed by overlaying the single molecule reads to obtain a high quality consensus sequence (19); Harris et al (20) have recently applied SBS at the single molecule level to sequence the M13 genome.…”

Section: Resultsmentioning

confidence: 99%

Sequencing by Cyclic Ligation and Cleavage (CycLiC) directly on a microarray captured template

Mir

Salata

et al. 2008

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

Next generation sequencing methods that can be applied to both the resequencing of whole genomes and to the selective resequencing of specific parts of genomes are needed. We describe (i) a massively scalable biochemistry, Cyclical Ligation and Cleavage (CycLiC) for contiguous base sequencing and (ii) apply it directly to a template captured on a microarray. CycLiC uses four color-coded DNA/RNA chimeric oligonucleotide libraries (OL) to extend a primer, a base at a time, along a template. The cycles comprise the steps: (i) ligation of OLs, (ii) identification of extended base by label detection, and (iii) cleavage to remove label/terminator and undetermined bases. For proof-of-principle, we show that the method conforms to design and that we can read contiguous bases of sequence correctly from a template captured by hybridization from solution to a microarray probe. The method is amenable to massive scale-up, miniaturization and automation. Implementation on a microarray format offers the potential for both selection and sequencing of a large number of genomic regions on a single platform. Because the method uses commonly available reagents it can be developed further by a community of users.

show abstract

Section: Resultsmentioning

confidence: 99%

Sequencing by Cyclic Ligation and Cleavage (CycLiC) directly on a microarray captured template

Mir

Salata

et al. 2008

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

show abstract

“…Besides RNA expression profiling, the technology paves the way for numerous further applications [90,91]. In particular, single molecule DNA sequencing comes into reach [18].…”

Section: Ultrasensitive Bioanalysismentioning

confidence: 99%

Single Molecule Bioanalysis

Hesse¹,

Schütz

2007

CPA

View full text Add to dashboard Cite

Beyond doubt, nanobioscience represents one of the most innovative concepts in current basic and applied research. Technological advances provided the essentials for this ongoing scientific revolution, by making possible the observation, investigation and manipulation of biomaterials down to single molecules. Time-resolved single molecule detection has become the method of choice to characterize structural transitions between different functional states of isolated biomolecules. The development of ultra-sensitive detection schemes also has a strong impact on bioanalysis, as the sensitivity of biochemical assays could be dramatically increased. Whenever the available amount of sample is the limiting factor for unambiguous diagnosis e.g. in medical diagnostics, bioanalytics with single molecule sensitivity can be expected to become even an enabling technology. In this review, we describe methodological requirements for ultra-sensitive detection, and point out major advantages, in particular the novel information content.

show abstract

“…Here, single molecule detection approaches can directly give information on the labeling stoichiometry of the sample and yield more precise results [10].…”

Section: Introductionmentioning

confidence: 99%

Single molecule fluorescence microscopy for ultra-sensitive RNA expression profiling

et al. 2007

View full text Add to dashboard Cite

We developed a microarray analysis platform for ultra-sensitive RNA expression profiling of minute samples. It utilizes a novel scanning system for single molecule fluorescence detection on cm 2 size samples in combination with specialized biochips, optimized for low autofluorescence and weak unspecific adsorption. 20 µg total RNA was extracted from 10 6 cells of a human keratinocyte cell line (HaCaT) and reversely transcribed in the presence of Alexa647-aha-dUTP. 1% of the resulting labeled cDNA was used for complex hybridization to a custom-made oligonucleotide microarray representing a set of 125 different genes. For low abundant genes, individual cDNA molecules hybridized to the microarray spots could be resolved. Single cDNA molecules hybridized to the chip surface appeared as diffraction limited features in the fluorescence images. The à trous wavelet method was utilized for localization and counting of the separated cDNA signals. Subsequently, the degree of labeling of the localized cDNA molecules was determined by brightness analysis for the different genes. Variations by factors up to 6 were found, which in conventional microarray analysis would result in a misrepresentation of the relative abundance of mRNAs.

show abstract

Ultrasensitive RNA profiling: Counting single molecules on microarrays

Cited by 18 publications

References 28 publications

Sequencing by Cyclic Ligation and Cleavage (CycLiC) directly on a microarray captured template

Sequencing by Cyclic Ligation and Cleavage (CycLiC) directly on a microarray captured template

Single Molecule Bioanalysis

Single molecule fluorescence microscopy for ultra-sensitive RNA expression profiling

Contact Info

Product

Resources

About