Since tag sequences can be introduced in the synthetic reporter DNA molecules, and several methods are available for detecting numerous DNA sequences simultaneously. Solution-phase PLA has been adapted for multiplex analyses of proteins with readout, either by quantitative PCR or using next generation DNA sequencing. Here we have combined PLA with readout via dual tag microarrays for parallel, sensitive detection of sets of proteins and protein-protein interactions. Using the DTM technique each microarray feature specifically detects and amplifies signals from reporter nucleic acid molecules that comprise two tag Z-VAD-FMK Caspase inhibitor sequence elements, representing the interacting proteins. We have previously used the DTM technique to measure cDNA levels as well as reporter molecules from proximity ligation and padlock probe assays, achieving a radically decreased risk of cross-hybridization compared to standard microarray approaches. Here we apply PLA with DTM readout to identify pairs of DNA tags, derived from the two PLA probes whose DNA strands have become joined by ligation upon coordinated binding to the same target protein or protein complex.

The DTM readout can be applied for analysis of PLA products reflecting protein levels and interactions both in liquid samples and for fixed tissue sections on slides, to provide measures of all binary combinations of PLA probes. The approach can be generalized to interrogate all interactions within larger sets of investigated proteins. It has previously been shown that dual color microarray analysis of proteins, where two samples are compared directly against each other, outperforms single color approaches when it comes to reproducibility and power of discrimination of the assay. We therefore designed a cassette connector oligonucleotide to facilitate sample barcoding for dual-color readout of results from analyses of pairs of samples. The combination of PLA with dual color DTM readout for multiplex detection of proteins and protein-protein interactions is illustrated in Figure 1. Between two samples in the same spot of a microarray using dual color readout.

PLA is performed either on immuneprecipitates of molecules of interest from cell lysates, or on cells fixed on a microscope slide, using a variant of methods previously described. Reaction products from individual samples are barcoded with a unique DNA sequence by interposing a short sample-specific sequence when pairs of PLA probes are joined by ligation. Thereafter the ligated reporter molecules from the two samples to be compared are pooled into one reaction tube and jointly amplified by PCR. The amplified reporter molecules are allowed to hybridize to the arrays. A ligase is subsequently added, allowing only the reporter with two barcode tags complementary to the array oligonucleotide to be ligated into circles. The circles will template localized DNA amplification by RCA on the microarray, as previously described. Detection oligonucleotides, specific for the two sample barcodes and labeled with Cy3 or Cy5, are added.