Hi. How do you make sure that the probe was successfully bound...

YL
YX L
Jun 6, 2021
protocol Protocol: Biochemical Pulldown of mRNAs and Long Noncoding RNAs from Cellular Lysates Coupled with Mass Spectrometry to Identify Protein Binding Partners
Hi. How do you make sure that the probe was successfully bound to targeted RNAs? What is the quality-check procedure? Looking forward to any reply. Thanks!
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Anca Flavia Savulescu Author Answered Jun 8, 2021

University of Cape Town

Dear YX L,

Thank you for your inquiry.

We use single molecule FISH probes from Biosearch and we plan them using the Biosearch probe design engine (https://www.biosearchtech.com/stellaris-designer).

In terms of target specificity concerns - typically, we plan sets of 24-48 probes per gene, at the highest masking level (level 5 - highest specificity). The Biosearch designer "checks" the specificity and cross reactivity of probes, and it should suggest probes that do not cross react (again, depending on the masking level you choose). As such, the specificity and accuracy is extremely high, as in single molecule FISH.

Otherwise, our protocol in terms of hybridization of the probes is based on the single molecule FISH protocol, which has been shown extensively in the literature to work (probes binding their targets) for a variety of genes.

Hope this addresses your concern? Please write back if it doesn't.

Thank you again for your inquiry.

Anca
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