RNA sequential probing of targets (SPOTs) is a newly developed method for mRNAs profiling, based on sequential Fluorescence In Situ Hybridization (seqFISH). SeqFIH is a technology that enables the identification of many RNA transcripts directly in single cells preserving spatial context.
To demonstrate the method “mRNA, extracted from mouse cells, was captured on a locked nucleic acid (LNA) poly(dT)-functionalized coverslip and then hybridized with a pool of 323,156 primary probes targeting the coding regions of 10,212 mRNAs with 28to 32 probes per gene”. Transcripts were labeled with fluorescent probes in sequential rounds of hybridization to read out the temporal barcode for each transcript. The temporal barcodes could then be decoded to uniquely identify the RNA transcripts present in the sample. A custom SecureSeal Flowcell, (RD478685-M;) manufactured by Grace Bio-Labs was applied to the functionalized poly(dT) coverslips. Ports allowed for easy introduction of fluorescence probes in hybridization buffer for sequential hybridization rounds and probe removal.
The current barcoding space is sufficient to encode the entire transcriptome. The large number of probes used per gene allows genome-level coverage and high specificity and reproducibility. RNAs SPOTs were demonstrated to be an accurate and low-cost alternative to sequencing.