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 DNA template generation by PCR 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.
