Journal: PLoS ONE
Article Title: Microarray Analysis of microRNA Expression during Axolotl Limb Regeneration
doi: 10.1371/journal.pone.0041804
Figure Lengend Snippet: A. Comparison between the human and axolotl miR-21 target sites in their Jagged1 genes. Nucleotide alignment of the miR-21 target site-containing region present in the 3′-UTR of the human Jagged1 ( Hsa-Jag1 , NM_000214) and the axolotl Jagged1 ( Amx-Jag1 , JF907581). The 22 bases comprising the target site for miR-21 are in yellow except for the 7 bases complementary to the miR-21 seed region (green color). Vertical bars (|) denote nucleotide identities between the two sequences. The numbers at both sides of the alignment are the nucleotide position on each 3′-UTR. B. In vitro luciferase assays testing the effect of miR-21 on the 3′-UTR of Amex-Jag1 in axolotl cells . Results are expressed as percent of co-reporter-normalized Renilla activity against reference vectors. Bars denote standard error of mean of the amount of independent assays. Student t- test was done and the obtained p- values determined that the Target of Hsa-miR-21 is a good positive control as biosensor for the activity of this microRNA ( p ≤0.005; two-tailed t- test). These results suggest that Amex-Jag1 may in fact be a target for miR-21 because significant differences were found in the Renilla signal recorded from cells electroporated with only the vector containing the 3′-UTR of Amex-Jag1 , versus the cells that were also electroporated with Pre-miR-21 *** (p≤0.005) but not Pre-miR30a. When the latter experiment was repeated with the mutant, seedless - Amex-Jag1- 3′-UTR, any sensitivity to exogenous miR-21 was lost.
Article Snippet: After running the RNA samples on a gel and confirming their integrity, 1.2 μg were sent to LC Sciences (Houston, TX) to be processed using their MicroRNA detection Microarray Service, and to be hybridized to a custom vertebrates chip (MRA-2001).
Techniques: Comparison, In Vitro, Luciferase, Activity Assay, Positive Control, Two Tailed Test, Plasmid Preparation, Mutagenesis
![Quantitative PCR validates miR-21 <t>microarray</t> results. Two first columns compare the averaged fold change between 17 dpa blastema (Bl) and stump (St) samples for LNA based qPCR assays (yellow bar), and for previous microarray data (red bar). Also, the individual fold changes between Bl and St for the three animals (biological replicates) are shown (blue bars). The relative miR-21 expression was calculated based on the efficiency corrected ΔΔCt method and normalized with miR-20a and miR-200b . The y-axis is a log scale. A fold change >1 indicates upregulation in Bl compared to St samples [ p ≤0.03 (qPCR), p ≤0.0001 (array); two-tailed t- test]. Inset , illustrates how non-isotopic Northern blot using digoxigenin-labeled LNAs against hsa-miR21 and hsa-U6 (control) are useful to detect the axolotl versions of these small RNAs. The axolotl miR-21 ( Amex-miR-21 ) is detected as a band of ∼20 nt which is over expressed in blastema when compared with stump and blood samples.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1534/pmc03441534/pmc03441534__pone.0041804.g003.jpg)
