Bioinformatics is a routine method for identifying miRNA target genes using free energy-based predictions and thermodynamic calculations to match similar sequences in databases to understand their binding affinities. Then, putative genes were predicted based on conserved regions of the 3’UTR sequences. Although some target genes of a small number of miRNAs have been successfully predicted, a large number of potential targets have been mispredicted due to imperfect pairings between the nucleotides of miRNAs and target mRNAs.
Furthermore, some true target sequences on the 3’UTR of a particular miRNA are extremely difficult to predict, as these target sequences may contain multiple elements that lack canonical seed pairings, thus requiring combinatorial binding of weak sites. Alternatively, microarray analysis has been used to compare relative expression levels of mRNA in the presence or absence of miRNA. While this approach has yielded at least 100-200 putative target genes of known miRNAs, it is cumbersome to apply, and variations in mRNA levels often make identifying true target genes difficult.
Bypassing bioinformatics and microarray methods, the researchers developed an experimental approach to search for target genes of known miRNAs. First, the researchers tagged the pre-miRNA with digoxin (DIG), which was then mixed with cell extracts. Endogenous dicers cleave this complex in vitro and produce mature miRNAs. Next, this DIG-tagged miRNA attaches to its target gene via the endogenous RNA-induced silencing complex (RISC). The miRNA-target mRNA mixture was then pulled down by anti-DIG antiserum.
To ultimately identify the target genes of a given miRNA, researchers clone all cDNAs from total mRNA, then pull them down for further DNA sequencing or clone them out by reverse transcriptase polymerase chain reaction (RT-PCR). In addition, to increase method certainty, they employed microarray analysis to analyze all mRNAs that were pulled down, further validating the predicted target genes of the tagged miRNA pull-down (LAMP) analysis system described here.
Microarray analysis was used by comparing the original wild-type pre-miR-1 probe with the original wild-type pre-miR-1 probe and the mutant (MT) pre-miR-1 probe, ug ca uaguaaagaaguauguau, where miR- The three substituted nucleotides at the 1 5′ end are underlined. Pre-miR-1 and mutated pre-miR-1 were labeled with DIG and mixed into cell extracts obtained from embryos at 24-48 hpf. miRNA/protein (miRNP)/mRNA complexes are pulled down by anti-DIG coated on agarose beads. Bound mRNA was isolated and then identified by microarray analysis.
Finally, the WT signal was normalized with the signal from MT (WT/MT) to quantify the target mRNA. Microarray analysis was performed using a single-color strategy. Normalize the WT signal with the signal of MT (WT/MT) to quantify the target mRNA. Microarray analysis was performed using a single-color strategy. Normalize the WT signal with the signal of MT (WT/MT) to quantify the target mRNA. Microarray analysis was performed using a single-color strategy.
This was demonstrated by using microarray analysis to compare the composition and levels of mRNA pulled down in the pool by miR-1 probes (WT) and mutated miR-1 probes (MT). Typically, levels of 28S or 18S are used to normalize the amount of RNA used for comparison. Compared to traditional microarray analysis, the LAMP detection system is relatively simple because it enables the acquisition of putative target mRNA complexes bound by DIG-tagged miRNAs from cell extracts. It is known to perform microarrays by overexpression or by knockdown/knockdown of specific miRNAs after comparing expression levels to those of wild-type embryos. However, in some cases, it should also be noted that the decrease or increase in gene expression levels displayed on the microarray may be caused by other mechanisms, including the influence of genes other than the target gene.
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