Is the v3.0 technology public now?
Yes! The in situ HCR v3.0 paper is now published. In situ HCR (v3.0) dramatically outperforms:

• in situ HCR v2.0 (tech development, protocols for 8 organisms)
• single-molecule HCR imaging (0.5 mm brain sections)
• quantitative HCR imaging (signal scales approximately linearly with target abundance)

The v3.0 technology provides automatic background suppression throughout the protocol, dramatically improving:

• Performance: high signal-to-background and quantitative precision.
• Ease-of-use: no probe set optimization for new targets.

The benefits of automatic background suppression are illustrated in Figure 3 of the v3.0 paper.

Should I switch to v3.0 now?
If you are already getting good results using v2.0 kits for your current project, there is no reason to change. However, if you are starting a new project and will be looking at new targets or doing exploratory studies in a new organism, we highly recommend switching to v3.0. The enhanced probe sets for v3.0 cost a little more but the difference in performance and robustness can be dramatic (see Figure 4 in the v3.0 paper).

What changes with v3.0?
The v3.0 protocol requires only two stages (detection and amplification) just as before. Also, v3.0 uses the same HCR amplifiers as v2.0, so there is no need to replace your amplifier stocks. The probe technology and buffers are different, however, and are not compatible between protocols, so each multiplexed experiment must standardize on either v2.0 or v3.0 for all targets.

How do I order v3.0 kits (or components)?
The web site has now been updated to provide a v3.0 ordering page.

Is in situ HCR v3.0 quantitative?
In situ HCR v3.0 supports three quantitative modes: 

• qHCR imaging: analog mRNA relative quantitation with subcellular resolution (overnight amplification, lower magnification); HCR signal is analog in the form of fluorescent voxel intensities that scale approximately linearly with the number of target molecules per voxel (see Figure 5 in the v3.0 paper).
• qHCR flow cytometry: analog mRNA relative quantitation for high-throughput single-cell analysis (overnight amplification); HCR signal is analog in the form of single-cell fluorescent intensities that scale approximately linearly with the number of target molecules per cell (see Figure 6 in the v3.0 paper).
• dHCR imaging: digital mRNA absolute quantitation (45-90 min amplification time, higher magnification); HCR signal is digital in the form of diffraction-limited dots representing individual target molecules (see Figure 7 in the v3.0 paper).

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

• qHCR imaging and qHCR flow cytometry: 20 or more split-initiator probe pairs per target mRNA; precision increases with probe set size.
• dHCR imaging: 30 or more split-initiator probe pairs per target mRNA (maximize probe set size based on length of target); fidelity increases with probe set size.

Did our name change?
Yes! After 14 years of molecular technology development in the Pierce Lab at Caltech, Dr. Harry Choi departed to found Molecular Instruments, Inc. (molecularinstruments.com), which will ramp up during 2018 and beyond to design, manufacture, and sell HCR imaging kits for academic research, biopharma R&D, and clinical diagnostics.

Dr. Maayan Schwarzkopf, who has been developing molecular technologies in the Pierce Lab since 2007, is the new Director of the non-profit academic resource at Caltech, which changed its name to Molecular Technologies (moleculartechnologies.org) to avoid namespace confusion.

Once MI becomes active, the v3.0 mRNA imaging kits will be available there instead of at MT. As it has since 2010, MT will continue developing and sharing next-generation programmable molecular technologies for biological research (we have many things in the works that we will be excited to share pre-publication as soon as they are validated in-house).

Do we like to see the latest and greatest images from your research?
Yes! We are always excited to see images from your research and hear about your latest discoveries so please keep sharing!

What comes in a v3.0 HCR kit?

• A validated or custom DNA probe set (default: 20 split-initiator probe pairs).
• A validated fluorophore-labeled DNA HCR amplifier.
• Hybridization, wash, and amplification buffers.

Can I omit one or more items from a kit?
Yes. Just select "None" for the scale of any item you want to omit.

What are the differences between Validated Kits and Custom Kits?
Validated Kit:

• Kit validated in situ by the Molecular Technologies Team.
• Serves as positive control.
• Note: due to high demand following the publication of the v3.0 paper, shipping times are now 1-4 weeks depending on our inventory and the details of your order. We thank you for your patience.

Custom Kit:

• Probe set synthesized from scratch.
• We aim to ship custom v3.0 kits within approximately 4 business days.
• Note: due to high demand following the publication of the v3.0 paper, shipping times are now 1-4 weeks depending on our inventory and the details of your order. We thank you for your patience.

What do I order for a multiplexed experiment?
Order one kit for each target mRNA. For each kit, choose a different amplifier (B1, B2, B3, B4, B5) and fluorophore (Alexa488, Alexa514, Alexa546, Alexa594, Alexa647).

Can I mix and match HCR amplifiers and Alexa Fluors?
Yes. You can order any of five DNA-HCR amplifiers (B1, B2, B3, B4, B5) with any of five fluorophores (488, 514, 546, 594, 647). Contact support to request an alternative fluorophore.

Can I order an HCR amplifier labeled with a custom fluorophore?
Yes. For an off-catalog fluorophore, select Sample: “generic” and Amplifier Scale: “3 nmol” and note the desired fluorophore in the “Comment” field. We will adjust the price (if needed) depending on the cost of the dye. If desired, you can request to have the dye distributed over more than one HCR amplifier (B1, B2, etc).

I've never used HCR – what’s the best way to get started?
Start with a validated v3.0 kit if possible (e.g., for a fluorescent protein mRNA). Otherwise, order a custom v3.0 kit for a high- or moderate-copy target (default: probe set with 20 split-intiator probe pairs).

How long does it take to perform in situ hybridization for five target mRNAs?
The same amount of time it takes for one target mRNA. We favor a 36-hour protocol that includes two overnight incubations, enabling the researcher to maintain a normal sleep schedule.

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). This can be a problem using standard probes (v2.0) since a “bad probe” that binds non-specifically in the sample will still be capable of triggering HCR. This is not a problem using split-initiator probes (v3.0) as probes only trigger HCR if they are colocalized by the target mRNA.
• NSA: non-specific amplification (HCR hairpins bind non-specifically).

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 using v3.0, something is wrong. Contact the Molecular Technologies Team.
• If the background is dominated by NSD using v2.0, test the probes individually to eliminate “bad probes” that are not selective for the target.
• If the background is dominated by NSA, something is wrong. NSA should typically be negligible relative to AF + NSD. Contact the Molecular Technologies Team.

Is Molecular Technologies a company?
No. Molecular Technologies is a non-profit academic resource within the Beckman Institute at Caltech.

How do I pay for a kit?

• Credit card (no transaction fee, no delay).
• Wire transfer or ACH ($35 transaction fee). For first-time customers, production begins upon receipt of payment. For repeat customers, please email your wire transfer or ACH receipt so that production can begin before the payment arrives.
• Caltech PTA (no transaction fee, no delay, Caltech users only).

My purchasing department requires a PO. Can I still buy a kit?
Yes. If your purchasing department requires a PO, you can save a PDF of your cart to provide as a quote (including a PO# in the reference field if desired). Your purchasing agent can then log in to your Molecular Technologies account, select the cart and proceed to payment, which must be made by credit card, wire transfer, or ACH.

What if I have other questions?
Please contact the Molecular Technologies Team.