Tips to Boost Performance in Your HCR™ RNA Imaging Experiments

Getting strong, clear signal in your HCR™ RNA imaging experiments sometimes requires a bit of fine-tuning. Whether you're using the HCR™ Gold or legacy HCR™ (v3.0) assay, small adjustments to your workflow can go a long way in improving signal intensity and overall performance.

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Below, we outline key parameters you can modify to enhance staining results and get the most out of your experiment.

Optimizing Performance in HCR™ Gold

HCR™ Gold features re-engineered probes with HiFi Probe architecture and built-in background suppression. While it performs well out of the box, there are a few tweaks you can explore for even better results: 

• Extend Incubation Times: Increasing both probe hybridization and amplification incubation times to overnight can sometimes help signal, especially in thicker samples. Note: extending beyond overnight has shown minimal added benefit.

• Upgrade to a Boosted Probe: Switching to a boosted probe design (including more binding sites) can elevate signal without requiring changes to your protocol.

• Consider HCR™ Pro: Still struggling with low signal? HCR™ Pro offers even greater sensitivity, making it ideal for low-abundance targets or highly autofluorescent samples. Learn more about HCR™ Pro here.

Optimizing Performance in HCR™ (v3.0) 

• Increase Probe Concentration: Are you still using a 4 nM probe concentration? If so, try increasing to 20 nM by preparing either (1) 2 µL of probe stock in 100 µL HCR™ Probe Hybridization Buffer or (2) 10 µL of probe stock in 500 µL HCR™ Probe Hybridization Buffer depending on your sample's reaction volume.

• Extend Incubation Times: As with Gold, incorporating overnight probe hybridization and amplification incubations can improve signal strength. Going beyond overnight is typically unnecessary.

• Upgrade to a Boosted Probe: If your RNA target sequence is long enough, consider ordering a Boosted probe to increase target site coverage.

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Dealing with autofluorescence? 

If background signal is interfering with your readout, check out our blog post on troubleshooting autofluorescence for initial guidance.

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Still not seeing the signal you need? 

If you've tried all the tactics above and are still seeing subpar performance, it's possible you've reached the sensitivity limit for your specific sample or target. While we may not have further tweaks to offer beyond what's covered here, we hope this guide has helped you get as close as possible to your experimental goals.