The read-out/read-in analysis framework enables bidirectional quantitative RNA discovery in an anatomical context.

Historical Approach

Historically, quantitative RNA analyses for vertebrates have sacrificed anatomical context, processing the sample using microdissection, dissociation, cell sorting, and/or homogenezation, and then quantifying the RNA via qPCR, RNA-Seq, flow cytometry, microarray analysis, or hybridization barcoding​.

HCR Approach

By quantifying RNA while preserving anatomical context, qHCR and dHCR imaging enable bidirectional quantitative discovery: ​​

Read-Out from anatomical space to expression space to discover quantitative co-expression relationships in selected regions of the specimen.

Read-In from expression space to anatomical space to discover those anatomical locations in which selected quantitative co-expression relationships occur.

Read-Out/Read-In Analysis Framework

qHCR and dHCR Quantitative Imaging Modes

Read-out/read-in can be performed using either quantitative HCR imaging mode: analog RNA relative quantitation with subcellular resolution (qHCR mode) or digital RNA absolute quantitation with single-molecule resolution (dHCR mode). See comparison  

Read-Out Example

The expression scatter plot below illustrates read-out from a whole-mount zebrafish embryo using qHCR imaging, providing quantitative snapshots of gene co-expression during somite formation and maturation.

Amplitude and Slope

Quantitative subcellular voxel intensities shaded by anatomical region reveal changes in expression ratio (slope) and abundance (amplitude) for mRNAs tpm3 and myod1 during somite formation (PSM) and maturation (S10→S9→S8→S7).

 RNA quantitation in an anatomical context

✓ qHCR imaging 

✓ dHCR imaging

Thick autofluorescent samples