Design and validation of BCR::ABL1 p210 SHERLOCK assay for CML. (A) Illustration of BCR::ABL1 p210 target-specific assay design strategy with fusion-specific RT-RPA primers and junction-specific crRNA guides. (B) LOD for isoform-specific BCR::ABL1 p210 RNA. (C) LOD for isoform-agnostic BCR::ABL1 RNA. Dilution series of K562 and KCL-22 RNA measuring LOD for (D) e13a2 RNA and (E) e14a2 RNA as a percentage of a fixed 10 ng of total RNA input. (F) Heat map illustrating background-subtracted fluorescence intensity for isoform-specific and isoform-agnostic CML BCR::ABL1 SHERLOCK using RNA extracted from patient samples. (G) Background-subtracted fluorescence from isoform-agnostic CML BCR::ABL1 SHERLOCK performed on diagnostic BCR::ABL1 RT-PCR–positive samples (n = 13) and control samples (n = 13). (H) Summary concordance of isoform-agnostic CML BCR::ABL1 SHERLOCK and BCR::ABL1 RT-PCR.
Figure 3.

Design and validation of BCR::ABL1 p210 SHERLOCK assay for CML. (A) Illustration of BCR::ABL1 p210 target-specific assay design strategy with fusion-specific RT-RPA primers and junction-specific crRNA guides. (B) LOD for isoform-specific BCR::ABL1 p210 RNA. (C) LOD for isoform-agnostic BCR::ABL1 RNA. Dilution series of K562 and KCL-22 RNA measuring LOD for (D) e13a2 RNA and (E) e14a2 RNA as a percentage of a fixed 10 ng of total RNA input. (F) Heat map illustrating background-subtracted fluorescence intensity for isoform-specific and isoform-agnostic CML BCR::ABL1 SHERLOCK using RNA extracted from patient samples. (G) Background-subtracted fluorescence from isoform-agnostic CML BCR::ABL1 SHERLOCK performed on diagnostic BCR::ABL1 RT-PCR–positive samples (n = 13) and control samples (n = 13). (H) Summary concordance of isoform-agnostic CML BCR::ABL1 SHERLOCK and BCR::ABL1 RT-PCR.

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