DTM readout of PLA on fixed cells.

<p><i>In situ</i> PLA (red) and immunofluorescence (green) detection of SMAD4 in <b>a</b>) cells transiently transfected with SMAD4 and <b>b</b>) wt cells having only endogenous levels of SMAD4. <b>c</b>) DTM readout of the ratios between SMAD4 from transfected cells and wt cells: transfected/wt (i), wt/transfected (a dye-swap experiment) (ii), transfected/transfected (iii), normalized against the ratio of beta-actin.</p

Detection of VEGF by PLA digital ASMD readout.

<p>Blue dots: detected signal; Diamond symbol: CV%.</p

Schematic illustration of PLA analysis with DTM readout.

<p><b>a</b>) The PLA probes, each carrying unique DNA sequences (A1, B1, …, N1, or A2, B2, …, N2) that code for their target antigens, are incubated with the samples to be examined. <b>b</b>) The oligonucleotides on pairs of PLA probes that have bound their targets in close proximity are ligated to give rise to reporter DNA strands. In the ligation step a sample barcode is introduced in the ligation product to allow for dual color comparisons of results for two samples in the same array spot. <b>c</b>) The ligation products are amplified by PCR and treated with DNA modifying enzymes to generate single stranded reporter molecules with barcodes identifying the targeted proteins at both ends. <b>d</b>) The reporter molecules are hybridized to oligonucleotides complementary to pairs of protein tags (e.g. N1N2), on a microarray, thereby allowing the reporter strands to be ligated into circles<b>. e</b>) The circularized reporter molecules finally template RCA, primed by the oligonucleotides on the array, and the RCA products are detected by hybridization with Cy3 or Cy5 labeled detection oligonucleotides. <b>f</b>) The Cy3 and Cy5 intensities are measured, and the ratios between the two colors are analyzed for each feature to detect differences in interaction patterns and protein abundances between the two samples.</p

Detection of p50, RelA, RelB, IκBα and GAPDH, and any pairwise interactions in cell lysates.

<p><b>a</b>) Cells were transfected with genes encoding proteins of the NFκB family; either p50, IκBα and RelB (sample I); or p50, IκBα and RelA (sample II). The two populations of cells were lysed and the PLA probes were added to the cell lysates in separate reactions. After generating the reporter molecules by ligation, the two samples were pooled to amplify the ligation products by PCR, followed by readout via DTM. <b>b</b>) The results are represented in a heat map where negative log₂ Cy5/Cy3 ratios (color coded as more green) indicate a higher abundance of proteins or protein interactions in sample I than in sample II, while positive log₂ Cy5/Cy3 ratios (color coded as more red) indicate the opposite situation.</p

Schematic illustration of protein detection by using PLA with digital ASMD readout.

<p>Schematic illustration of protein detection by using PLA with digital ASMD readout.</p

(A) Standard curves for IL6 detection by PLA followed by digital ASMD readout (blue dots) or realtime PCR based readout (red dots).

<p>Error bar = ±1SD. (B) Comparison of the CV% between the two readout strategies (blue dots: digital ASMD readout; red dots: realtime PCR readout).</p

Opportunities for Sensitive Plasma Proteome Analysis

Despite great interest, investments, and efforts, the ongoing search for plasma protein biomarkers for disease so far has come up surprisingly empty-handed. Although discovery programs have revealed large numbers of biomarker candidates, the clinical utility has been validated for only a very small number of these. While this disappointing state of affairs may suggest that plasma protein biomarkers have little more to offer for diagnostics, we take the perspective that experimental conditions might not have been optimal and that analyses will be required that offer far greater sensitivity than currently available, in terms of numbers of molecules needed for unambiguous detection. Accordingly, techniques are needed to search deep and wide for protein biomarker candidates. The requirements and feasibility of such assays will be discussed

Comparison between confocal microscopy and the dedicated instrument for detection of RCPs.

<p>The quantitative response of the same dilution series of EC DNA was measured using the dedicated instrument as well as the confocal setup used in Jarvius <i>et al</i> 2006. Filled symbols: dedicated instrument, open symbols: Zeiss 510 Meta confocal microscope.</p

Detection scheme of the bio-monitoring system.

<p>A) Collection of environmental samples is realized using the Airborne Sample Analysis Platform (ASAP) equipment. Aerosolized particulates stick to a filter, and the content is extracted and analyzed. In contrast to protein detection, detection of nucleic acids requires preparation of the filter content prior to exposure to the molecular procedures. The molecular procedures detect the target molecules using dedicated probes and reacted probes are then amplified. The amplification products are analyzed using a dedicated prototype instrument. B) The molecular procedures of DNA (left) and protein (right) detection. Detection of nucleic acids is achieved by padlock probes that are specifically circularized if correctly hybridized to the correct target in the presence of DNA ligase. Padlock and capture probes are added to the samples along with DNA ligase (5 min). Reacted padlock probes are captured on magnetic beads and excess probes are eliminated by washing (3 min). Detection of proteins is initiated by capture of the target protein using magnetic beads equipped with antibodies. The addition of a pair of PLA probes, which are antibodies with attached oligonucleotides (3 min), forms a DNA circle guided by two connector oligonucletides, and a DNA ligase (5 min). Unreacted probes are eliminated by washing the circles, and from this step the magnetic beads are treated identically in both the genetic and PLA assays. A first RCA is initiated either by an extra primer or the target itself on the beads to replicate the DNA circles (11 min). The products are then restriction digested (2 min), and the monomers are collected. The monomers can then bind head-to-tail to the excess amount of replication oligonucleotides and formed new DNA circles. The new circles are amplified and labeled with fluorescence-tagged detection probes. The ligation, amplification and labeling are performed in one reaction (8 min). The RCPs are therefore ready for analysis in the detection instrument.</p

Sequences of the oligonucleotides used in the study. Underlined nucleotides are locked nucleic acids [17].

<p>Nucleotides in italic style are 2′OMe-RNA. The NRP is furthermore labeled by biotin in the 3′end.</p