hello, actually i'm working on transcriptional analysis of primary microRNA-223 in chicken and i'm trying to find the TSS and determine the different spliced transcripts. I've tried nested PCR using different primers in different positions but no amplicon were obtained.the bioinformatic analysis didn't help me a lot because the gene of this microRNA is not annotated except the precurseur microRNA. Moreover, i can't rid of the genomic contamination in my samples even after DNASE I treatment.
Could anyone help me please,
You could try to deplete Drosha with a siRNA to increase the half life of the pri-microRNA, it might be easier to detect then.
First, keep in mind that what you are doing is very difficult. I did this myself for miR-181-1 and miR 181-2 in humans.
http://www.ncbi.nlm.nih.gov/pubmed/25410655
You may not need a Drosha knockdown to see the pri-microRNA. With some miR genes, the predominant transcript is an unspliced transcript and so you can map where the beginning of the gene is with qPCR as I did in my paper. The contamination you see is possibly the unspliced gene transcript, like I saw and others have seen:
http://www.ncbi.nlm.nih.gov/pubmed/24073292
Let me know if you need additional help.
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