Detection And Analysis Of Abortive Transcripts Associated With Muc16 Gene In Cancer Cells

Abortive transcript（AT）refers to a class of special non-coding RNAs in the range of 2-19nt in length produced AT the abortive initiation（AI）stage.Generally,AI occurs within the organism.RNA polymerase（RNAP）in AI stage can’t escape from promoter regions,but again,repeatedly synthetic<10nt RNA fragments,until the synthesis>10nt pieces of RNA fragment,RNAP can give play to the role of the real transcription.AT high frequency synthesis and release are bound to have important biological significance.However,the existing problem is that it is difficult to carry out qualitative and quantitative analysis when AT fragments are concentrated below 10nt.In order to solve this problem,this study used base stacking hybridization and toehold-mediated strand replacement reaction to effectively realize quantitative detection of short fragment RNA.CA125 has been identified as a biomarker for a variety of cancers.muc16,a gene that expresses CA125 protein,is bound to release a large amount of relevant AT in the AI stage.Therefore,muc16 gene-related AT may be a potential tumor marker that can be used for the detection of cervical cancer and other cancers.This study will provide technical support for the development of muc16-related AT,as well as research ideas and technical support for the development of other genes of AT as a new biomarker for early diagnosis of disease.The main research contents and conclusions are as follows:（1）Effect of short RNA on base accumulation hybridizationIt was found that when the final concentration of Mg²⁺was above 6mM in BSH-ABC mode,the fluorescence signals of B_BHQ1 and C_FAM tended to be stable,indicating that the ssDNA B was bound to the A strand in A under the base accumulation energy provided by AC.In a certain concentration range(B_BHQ1:AC=0.25:1,0.5:1,0.75:1,1:1),B_BHQ1 was negatively correlated with the fluorescence signal.However,in the case of excessive B_BHQ1(B_BHQ1:AC=2:1,5:1),the fluorescence signal tended to be stable,indicating that the binding efficiency of short strand was closely related to the concentration of long strand scaffold（AC）.Because the BSH-ABC model has problems in detecting ssDNAs:the naturally existing ssDNAs are not modified by fluorescent groups,in this view,we designed the ABCD model suitable for the detection of natural ssDNAs,which contains two ssDNA:C and D.In addition,in the BSH-ABCD mode,when the final concentration of Mg²⁺was above 3mM,the fluorescence signals of B_FAM and A_BHQ1·LNAHQ1·LNA tended to be stable.The concentration of magnesium ions used in the BSH-ABCD mode was lower than that in the BSH-ABC mode,possibly due to the increase of LNA.In addition,we studied the effect of the concentration gradient of D_8nt on the fluorescence signal of BSH-ABCD,and found that there was a negative linear correlation between the concentration and fluorescence signal.This study can realize the quantitative detection of ssDNA.（2）Short strand nucleic acid interferes with chain replacement mediated by ToeholdWe discussed the effects of different types of accumulation patterns,cations of different concentrations,and invasion strand of different lengths（E）on TMSD.Finally,the detection scheme as shown in 3-3 d was determined,and the final concentration of Mg²⁺was determined to be 5mM,and the invasion strand was determined to be 27nt(E_I5+22).Based on the above conditions,the standard curve between D_ATT concentration and TMSD initial reaction rate was established,and it was found that the linear relationship between D_AT concentration and TMSD initial reaction rate was negatively correlated.Based on the results of the above studies,the quantitative detection of ssDNA can also be achieved.（3）Quantitative analysis of intracellular abortive transcriptsIn the study of（2）and（3）,we preliminarily discussed the relationship between D_AT concentration and the stable binding of BSH-ABCD mode and the initial reaction rate of TMSD with synthetic short stranded RNA(D_AT),and constructed the standard curve,both of which were negatively correlated with the concentration of ssDNA(D_AT).We selected HEK-293,A549 and Hela as the control group and the experimental group,respectively,and extracted small RNA.According to the standard curves constructed according to the initial reaction rates of D_AT/BSH-ABCD and D_AT/TMSD,it was found after quantitative calculation that the trend of detecting AT content by the two methods was consistent.In summary,this study has successfully established two methods for the detection of ssDNA,and can conduct quantitative detection,providing a feasible scheme for the further develop at as a new biomarker and study of the biological function of AT.