Research Proposal on the Expression of MicroRNA in the Evolution of an Insect

Research Proposal on the Expression of MicroRNA in the Evolution of an Insect
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Introduction
MicroRNA (miRNA) is significant in the regulation of the posttranscriptional gene expression. MicroRNAs are short endogenous RNA sequences and they facilitate translational repression of the mRNAs. The miRNAs have roles in most biological processes like development and signalling viral infections. In multicellular organisms, they are ubiquitous whereas, they are absent in organisms that are single-celled. This shows a role in multicellularity (Casey et al, 2015). In insects, miRNA facilitate in the regulation of development aspects (patterning of the body and cell differentiation) hence, the changes in the functional features of the insects are linked to the evolution of body plans and phenotypic variation with related species (Santana). This has then sparked interest in familiarizing with the evolution of the miRNA expression. MicroRNA is used as a rare genome change character in estimating phylogeny through taking note of gains and losses. The short length has however limited the strands perceived utility in sequence based phylogenetic inference. With the use of reference taxa, phylogenetic relationships are demonstrated in the miRNA sequence, quantitative instead of qualitative phylogenetic analysis (Kenny et al, 2015; Santana).
Experimental plan
Materials and methods
Identification of miRNA and sequence recovery
The complete components of miRNA of the beetles T.castaneum would be used to identify the number of miRNA. Each miRNA would be extracted from the genome of the beetles. JMODEL TEST would be utilized to come up with the best fitted model of nucleotide substitution and the GTR + 1 +4G model (Kenny et al, 2015).
Generation and sequencing
The beetles would be cultured at 28 degrees Celsius, then RNA would be extracted from embryos and adults that have the miRVana miRNA isolation kit (Marco et al, 2010). The molecules that would be shorter than 40 nucleotides would be preferred and selected using the flash PAGE fractionator and then they would be purified with the flashPAGE reaction clean up kit. Libraries that would contain different embryonic stages will be erected having barcodes and SOLiD Small RNA Expression Kit. Small RNA, according to the size selection would be litigated to sequencing adapters. Reverse transcription would be carried out together with RNaseH digestion in order to have the cDNA libraries (Berezikov, 2010) The sample quantity of SOLiD sequencing would be met by further amplification of the cDNA libraries through utilizing different sets of barcodes via 15 and 18 cycles of polymerase chain reaction. The final products that would range from 105 to 150 bp would be purified (Kenny et al, 2013).
Detection of transcribed miRNAs
The reading of the SOLiD sequencing would be noted on the length of the nucleotides. It would be expected for putative miRNA mature sequence to be detected in the SOLiD run and it should have fragments of what was linked during the sequencing process (Marco et al, 2013). All the reading would be mapped to the annotated Tribolium rRNAs and the transfer tRNAs through the use of Bowtie. This will allow a mismatch between t...