The overexpression of mir-218 miRNA can be determined by the use of many different techniques.
One popular method would be quantitative real-time PCR, which starts with total RNA being extracted from a tissue sample by TRIZOL reagent REF, followed by reverse transcription with stem-loop-specific primers. The stem refers to a region complimentary to a known sequence on the 3’ end of the miRNA and the loop refers to a universal primer-binding sequence. The cDNA product then acts as a template for qRT-PCR along with mir-218 specific primers and a second universal primer (TaqMan PCR, Applied Biosystems). REF PCR results obtained from both normal and tumorous samples can then be compared to analyse differences in the mir-218 expression levels. Another method is
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After total RNA is isolated from cells/tissue, the RNAs are separated by electrophoresis and then transferred from the gel onto a membrane where they are fixed REF. The membrane containing the rna is then treated with a oligonucleotide probe, with an increased sensitivity required for effective mirna detection REF. This probe will contain a sequence complimentary to mir-218, allowing it to bind/hybridize to the specific fragment on the membrane. As well as containing a fluorescent/radioactive label to allow visualisation of the detected mirna. REF For analyse of mir-218 expression, PCR seems to be the superior method over northern blotting for many reasons. PCR, especially with the use of stem-loop primers avoids obtaining cDNA from non-specific targets, producing a more reliable result whereas Northern procedure is, in general, less sensitive. PCR also requires very