Hi everyone,
I have cloned precursor sequence of a miRNA (say mIR1) in Arabidopsis and generated three independent, homozygous transgenic lines in Arabidopsis. When I performed qRT-PCR of these lines to determine the expression level of precursor miRNA (pre-miRNA), I observed 6-, 5-, and 12-fold change when compared with wild-type Arabidopsis. Next, I did stem-loop qRT PCR to determine the level of mature miRNAs in these lines. Unfortunately, there is no change in mature miRNA observed. I designed the stem-loop primers according to Varkonyi-Gasic and Hellens, 2011. My reaction conditions were 95◦C for 2 min, followed by 40
cycles of 95◦C for 5 s and 60◦C for 10 s.
Have anybody faced similar problem! Any help/ suggestion will be highly appreciated!
Thanks
Hello Swati Megha,
Are you sure that your microRNA is not transcribed from the opposite strand? If so, you may detect the pre-miR, but the transcribed pre-cursor is not capable of forming proper stem-loop structure or be recognized by Dicer enzyme. Please be sure that you have cloned the pre-cursor in a proper direction in expression vector.
Besides, when the endogenous expression level of mlR1 is high, you cannot trace the overexpression efficiency.
Another solution: Try your experiment in other cell lines of Arabidopsis. If the miRNA is evolutionary conserved, you can overexpress it in othe plants and trace the processing of this miRNA to produce mature form.
Good luck
173
Hi Swati
Some useful references in attachment 1
https://www.ncbi.nlm.nih.gov/pubmed/25807486
6
https://onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2008.06624.x/full
7
https://biosignaling.biomedcentral.com/articles/10.1186/1478-811X-7-18
9
https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1006753
13
https://journal.frontiersin.org/article/10.3389/fpls.2017.00018/full
One possibility is that your PCR is not working correctly for whatever reason. I will instead do a Nothern blot to quantify mature and precursor miRNA. If this show the same as PCR then this will mean that the precursor is stuck in the nucleus or is not a good substrate for dicer.
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