4 Notes
- Due to the multiple tandem repeats, the plasmid should be
amplified inEscherichia coliMAX Efficiency Stbl2 (Thermo
Fisher) at 30C to minimize recombination, which results in
sequence deletion and/or rearrangement. - d2EGFP is a destabilized protein derived from EGFP and has a
shorter intracellular lifetime than EGFP. Genes encoding
EGFP or other proteins can also be used, provided that the
protein expression can easily be detected at the single-cell level
and that the mRNA sequence is unique in the cell type of
interest to allow for the design of specific smFISH probes for
single-molecule RNA detection. - Avoid centrifugation at speeds> 5000 gto avoid the RBMBs
drying on the membrane. - To achieve high transfection efficiency and cell viability, it is
important to ensure that cells are not>70% confluent prior to
microporation. - Enough time should be given for trypsinization to ensure all of
the cells are detached from the surface. Avoid vigorously pipet-
ting the cells.
Fig. 3Quantifying single RNA transcripts using RBMBs and smFISH. Five hours following microporation of
HT1080-d2EGFP-96mer cells with 0.05–4.8μM of RBMBs, the cells were fixed and permeabilized and
smFISH was performed to assess the accuracy of MBs for detecting single RNA transcripts. (a) A custom
Matlab program was written to analyze the percentage of MB signals that were colocalized with smFISH
signals (open circles) and the percentage of smFISH signals that were colocalized with MB signals (black
diamonds) in individual cells. Data represent meanstandard deviation of at least 20 cells. (b) RNA copy
number per cell reported by smFISH and RBMBs (0.8μM) are in good agreement (Reproduced from [6] with
permission from Oxford University Press)
Ratiometric Bimolecular Beacons 239