Sensitive Quantification Of Messenger RNA With A Real-Time Ligase Chain Reaction

Replication, transcription and translation are basic life processes. m RNA, transcribed from DNA, translates into peptide at the ribosome. Peptide folded into protein. Protein closely relates to the biochemical pathways and pathogenic mechanism, so analysis of m RNA is important in life processes and disease diagnosis fields.Polymerase chain reaction(PCR) is a common method in detection of m RNA, but it requires reverse transcription of m RNA, and can not effectively identify single nucleotide differences. Ligase chain reaction(LCR) is a PCR comparable method for nucleic acid amplification. It can be distinguish single nucleotide differences very well. However, it needs electrophoretic separation for detecting the product, and the efficency is very low for jointing RNA-templated DNA probes.By using the ribonucleotide-modified DNA probe, we have realized the efficient ligation of DNA probes templated by the m RNA target. Through monitoring the fluorescence intensity of LCR products at appropriate temperature, a real-time LCR-based m RNA assay is developed. The proposed m RNA assay does not require a reverse transcription step before LCR and the separation and detection steps after LCR, thus, simplifies the m RNA detection procedure. The SG fluorescence dye, rather than fluorophore-labeled DNA probes, is utilized for the real-time measurements, significantly reducing the assay cost. The proposed m RNA assay also exhibits the advantages of high specificity to discriminate one-base difference between m RNAs and high sensitivity to quantify m RNA target in the concentration range from 1 fmol/L to 100 pmol/L. The preliminary test results have shown that the proposed m RNA assay can be well applied to accurate quantification of m RNA in very small amounts of total RNA samples. Therefore, the proposed m RNA assay should be of value in both research and diagnostics by using m RNA as the biomarker.