We use 250-1000bp long DIG- or FITC-labeled mRNA probes for ISH. These probes are made by in vitro transcription (IVT) kits and a gene specific DNA template.
We can make both template and labeled probes for you, but it is more cost-effective for you if you can provide the DNA template and then let us do the IVT.
DNA template generation: the easiest way to make the template is by PCR or cloning. For PCR instructions view our or talk to the core director by email at atc-rnacore@bcm.edu. Make sure to include T3 and T7 RNA polymerase binding sites at the flanking ends of your PCR product.
A double stranded DNA template can also be purchased either from companies that sell DNA fragments (approx. $100-250 per fragment depending on size and amount), or from the core ($150-200). If purchasing from a company, make sure you include the T3 and T7 RNA polymerase binding sites properly. Ask the core director by email at atc-rnacore@bcm.edu if you are unsure how to do this.
The core has a few thousand probes already made – send us an email and we’ll check if we have a probe for your gene. If we do, we can use our probes for your experiment for a small fee per slide.
DIG-labeled probes are more stable than FITC-labeled ones (6+ years vs. ~2 years shelf-life). We recommend making DIG-labeled probes most of the time and only making FITC-labeled when you need to perform a double ISH together with a DIG-labeled probe.
If you’re interested in microRNA (miRNA) you will need to purchase probes from Exiqon/Qiagen. miRNAs are too short for our normal protocol so you will need LNA-probes from this company. Email the core to get instructions for suitable probe labels at atc-rnacore@bcm.edu.
