Monitoring p21 mRNA expression in living cell based on molecular beacon fluorescence increasing rate

Abstract

Studying the expression level of mRNA in living cells will offer tremendous opportunities for advancement in cell biology research, disease diagnostics, and drug discovery. In this paper, a molecular beacon (MB) specific for the important tumor suppressor gene p21 has been designed and synthesized. The fluorescence signal was detected in real-time after the MB entered the cytoplasm of nasopharyngeal carcinoma cells. After injecting the p21MB into nasopharyngeal carcinoma cell and p33-transfected nasopharyngeal carcinoma cell, the consistent increase of fluorescent signal intensity was detected in both cell lines, and maximum fluorescence intensity achieved in about 15 min. In about 4 min following microinjection, the fluorescence increasing rate was significantly different between these two cell lines, which indicate the different p21 mRNA expression levels. The results obtained in the real-time detection were also validated by RT-PCR. Analysis of the initial fluorescence increasing rate can efficiently reduce the side effect of enzyme and improve the accuracy in living cell mRNA detection. molecular beacon, p21 mRNA, nasopharyngeal carcinoma cell, living cell imaging, fluorescence increasing rate Cell's structural and functional characteristics are dependent on the specific complement of genes expresses. Many cancers and cardiovascular diseases are associated with changes in particular genes' structure or abnormal expression level of particular genes [1 -3] . Real-time monitoring the dynamics of mRNA expression can offer tremendous opportunities for advancement in cell biology, molecular biology, medical diagnostics, and drug discovery [4] . It has become clear that a chemistry reaction in a test tube would have different thermodynamic and kinetic properties of the same reaction in a living cell [5,6] . Current RNA analysis techniques, including RT-PCR, Northern-Blot, and in situ hybridization [7][8][9][10][11] , can provide gene expression information by studying purified RNA obtained from cell lysate samples or by studying RNA in fixed cells. But these methods cannot visualize the expression level of specific RNAs in living cells in real-time. Therefore, mRNA has to be observed in real-time and in its native environment with high sensitivity [6] . However, it is difficult to study mRNA in living cells for the complicated cellular environments.MBs are dual-labeled single-strand oligonucleotide probes, with a fluorophore at one end and a quencher at the other end [12,13] . In the absence of a complementary target, the stem-loop structure keeps these two moieties in close proximity, causing the fluorescence of the fluorophore to be quenched. In the presence of target nucleic acid sequence, the loop region forms a hybrid and opens the stem. The spatial separation of the fluorophore and the quencher leads to the restoration of fluorescence emission from the fluorophore. MBs can report the presence of specific nucleic acids with high sensitivity and excellent specificity, and have been

Molecular Beacons: A Novel Optical Diagnostic Tool

“…The results showed that p21 mRNA is inhibited by silencing p53 in CNE2 cells, while it is upregulated in MCF-7 cells by ING1 transfection, the influence of 5-fluorouracil (5-FU) treatment on p21 mRNA of MCF-7 cells is time and concentrationdependent manner (Liu et al 2010a). After injecting the p21 MB into nasopharyngeal carcinoma cell and p33transfected nasopharyngeal carcinoma cell, the consistent increase of fluorescent signal intensity was detected in both cell lines, and the fluorescence increasing rate was significantly different between these two cell lines, which indicate the different p21 mRNA expression levels (Tang et al 2008). These results demonstrated that the new method with the advantages of high specificity, fast speed and simplicity can be widely applied for p21 mRNA detection in different samples.…”

“…(2013) 61:139-148 signal leads to a substantially increased signal-to-background ratio as compared with that seen in single MB assays and enables discrimination between fluorescence due to the specific probe/target hybridization and a variety of possible false-positive events. Tang et al (2008) used a phosphorothioate-modified MB detecting green fluorescent protein (GFP) mRNA in COS-7 cell and GFP-transfected COS-7 cell in real time based on living cell imaging method. The detection shows the advantages of MB, such as, excellent selectivity, high sensitivity, and no separation detection.…”

Detection of mRNA in living cells by double-stranded locked nucleic acid probes

“…Cells can function normally in the experiments and the results are comparable to reverse transcription PCR. 34, 35 However, the microinjection technique could be invasive, slow, and labor intensive. To address the requirement of single-cell delivery, we explored delivery of dsLNA probes by photothermal nanoblade, which has shown to have high efficiency and cell viability.…”

Protein analysis based on molecular beacon probes and biofunctionalized nanoparticles

“…The thermodynamic stability of the hairpin structure, the highly efficient intrinsic signal switching, and the possibility of using a variety of fluorophores make MBs exceptional biomolecular analysis probes with excellent sensitivity, selectivity and real-time detection capability. MBs are therefore used for a variety of applications, such as DNA and RNA detection [21][22][23][24][25][26][27][28][29][30], monitoring of living systems [31][32][33][34][35][36], investigation of enzymatic processes [37][38][39][40][41][42][43][44][45], design of biosensors [46][47][48][49][50], study of protein/DNA interactions [51][52][53][54][55][56][57], and the fabrication of biochips [58,59]. Here, the scope of this review is limited to the fundamental aspects of MB's principles, advantages and the recent applications of MBs in protein recognition and protein/DNA interaction studies.…”