Cloning and sequencing of microRNA-17 and -18a genes from HepG2 cancer cell line / Nazihah Abd Rahman

miRNA is a small non-coding RNA region that is not translated to protein. This region usually involved in the regulation and expression the cell. Many studies were done on the miRNA and showed high correlation with certain cancer disease such as liver cancer. The aim of the study is to clone the miRNA -17 -l 8a of HepG2 cancer cell line in a vector. Initially, the region was amplified using pair of primer designed based on the sequences of the miRNA -17 -18a region fron GenBank. Optimum annealing temperature obtained from the PCR optimization was 51 °c. The PCR product was then ligated into the plasmid based on TA cloning method. The presence of insert and plasmid into the competent cell was confirmed using blue white screening method. Besides that, PCR cloning using Ml3 universal primer was done to further prove the presence of insert in the plasmid vector. Nevertheless, based on the result of sequencing, the inserted region was not the desired region as it did not aligned with the sequence of miRNAl 7 and 18a from NCBI. This might due to the unspecific binding of the primer pair designed earlier.