FAQ

What comes in an HCR v3.0 bundle?

  • HCR probe set

  • HCR amplifier (validated design, QC-tested synthesis)

  • HCR buffers: probe hybridization buffer, probe wash buffer, amplification buffer

 

Can I order a subset of these components?
Yes! You can order bundles containing any subset of the above components. 

What do I order for a multiplexed experiment?
Choose a different HCR amplifier (B1, B2, …) for each target RNA that will be imaged in the same sample (for example, amplifier B1 for target 1, amplifier B2 for target 2, …). Choose a different fluorophore label (Alexa Fluor® 647, Alexa Fluor® 594, …) for each HCR amplifier that will be imaged in the same sample (e.g., B1-647, B2-594, …).

What probe set size do you recommend for v3.0?

  • qHCR imaging and qHCR northern blotting: 20 or more split-initiator probe pairs per target RNA (as permitted by target length); signal-to-background and precision increase with probe set size.

  • qHCR flow cytometry: 30 or more split-initiator probe pairs per target RNA (maximize probe set size based on length of target); signal-to-background and precision increase with probe set size.

  • dHCR imaging: 30 or more split-initiator probe pairs per target RNA (maximize probe set size based on length of target); fidelity increases with probe set size.

 

I've never used HCR – what’s the best way to get started?
Please see our Protocols, which provide a starting point for working in diverse organisms. Start with a positive control probe set if one is available for your sample type (e.g., a fluorescent protein mRNA). Otherwise, order a custom probe set for a high- or moderate-copy target.

I am getting great results with an HCR probe set that I previously ordered from moleculartechnologies.org – can I order more of the exact same probe set from Molecular Instruments™? 

Yes! Please include a note in the Comment field of your order (include your previous Order # if available).

 

How do I order and pay for HCR bundles?

  • Place an order for one or more bundles at the Molecular Instruments™ online store.

  • You will receive an email quote which you can use to upload your purchase order

  • Your institution will receive an email invoice at time of shipment

  • Payment is due by ACH or wire transfer within 30 days of the invoice date 

Can I cancel my order? 

Yes! You can cancel your order up until the time your purchase order is accepted. Please contact the Molecular Instruments Team (support@molecularinstruments.com).

 

Do you offer academic discounts? 

Yes! If your organization is a university, government lab, or non-profit research institute, you are eligible for academic pricing.

 

What are your estimated shipping times? 

Upon receiving a valid purchase order, we aim to ship as follows:

  • HCR probe set: within 1 business day

  • Custom HCR probe set: within 5 business days 

  • HCR amplifier: within 1 business day

  • Custom-labeled HCR amplifier: within 10 business days

  • HCR buffers: within 1 business day

 

Can I order an HCR amplifier labeled with a custom fluorophore? 

Yes. Choose Custom in the Amplifier Label pulldown and enter the name of the fluorophore (currently any of the Alex Fluor® dyes or Cyanine 7 or 7.5). Custom labeling requires a minimum scale of 5 nmol. The 5 nmol can be spread across up to three HCR amplifiers (e.g., B1-Cyanine 7, B2-Cyanine 7, B3-Cyanine 7), with a minimum of 1 nmol per amplifier (include a comment describing your custom label order in the Comment field). 

 

What are the benefits of automatic background suppression?
HCR v3.0 provides automatic background suppression throughout the protocol, ensuring that reagents will not generate amplified background even if they bind non-specifically within the sample.

 

Automatic background suppression dramatically enhances:

  • Performance: signal-to-background, qHCR precision, and dHCR fidelity.

  • Ease-of-use: no probe set optimization for new targets and organisms.

 

What causes background?
Background arises from any of three sources: 

  • AF: autofluorescence (inherent fluorescence of the fixed sample).

  • NSD: non-specific detection (probes bind non-specifically and are subsequently amplified). HCR v3.0 automatically suppresses NSD by using split-initiator probes that only trigger HCR if both probes within a pair are colocalized by the target mRNA. 

  • NSA: non-specific amplification (HCR hairpins bind non-specifically). HCR v3.0 automatically suppresses NSA by using kinetically trapped HCR hairpins that do not trigger HCR if they bind non-specifically in the sample.

 

How do I check autofluorescence (AF)?
Perform the standard in situ HCR protocol but leave out the probe set and amplifiers.

 

How do I check for non-specific detection (NSD)? 
In an embryo, compare staining to a reference expression pattern (if available). In cells, test for absence of signal in a knockout strain (if available). Otherwise, perform a redundant detection experiment by detecting the target with two probe sets in two channels and checking for correlation of the staining in the two channels (Choi et al., 2010, 2014, 2016, 2018). 

 

How do I check for non-specific amplification (NSA)?
Perform the standard in situ HCR protocol but leave out the probe set.

How do I increase the signal-to-background ratio?

  • If the background is dominated by AF, increase the number of probes in the probe set. 

  • If the background is dominated by NSD or NSA, something is wrong. These contributions should be negligible using HCR v3.0, which provides automatic background suppression throughout the protocol. Please contact the Molecular Instruments Team (support@molecularinstruments.com). 

Do you like to see my images?
Absolutely! We are always excited to see images from your research and to hear about your latest discoveries! 

What if I have other questions? 
Please contact the Molecular Instruments Team (support@molecularinstruments.com). 

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