Hello all,
I am fairly new to the world of RNA and sncRNA, so I appreciate any input. I am currently trying to amplify miRNA sequences with primers that I generated using the miRprimer algorithm in a multiplex RT-PCR. I have an exoRNEasy kit from Qiagen that suggests their c. elegans miRNA spike in control to be used during RNA extraction. However, the miRNA is to be amplified using their locked nucleic acid primers, which I am to understand is a difficult template for DNA polymerase. The melting temperature of the spike in control miRNA would be different, but shouldn't be an issue if I optimize with a gradient PCR. I'm wondering if there's a simpler method, such as ordering a c. elegans spike in control elsewhere and testing primers produced by miRprimer?
Thanks!
Hello Hannah Hipkiss
I would like to provide two inputs regarding your discussion:
- LNA primers basically can work in low amounts of DNA polymerase. Hence there is no difficulties (see PDF attached for more details), only a good polymerase selection is required.
- If you want to go for an alternative miRNA control (some other c.elegans based one instead of cel-miR-39, I can suggest you to cel-miR-54 or cel-miR-54-3p as controls from ThermoFisher (link given below), which are excellent alternatives for the Qiagen one. You can refer to articles based on cel-miR-54 or cel-miR-54-3p for further knowledge and applicability before use.
https://www.thermofisher.com/order/genome-database/details/microrna/001361?CID=&ICID=&keyword=cel-mir-54-5p&configurator=false&subtype=mirna_non_advanced#more-information-section
If any further queries, feel free to ask, or message me!
Cheers!
Please follow the link
Article Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers
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