Universal method for rapidly quantifying RNA residue in DNA product

Abstract

The invention discloses a universal method for rapidly quantifying RNA residues in a DNA product. According to the method, RNA is quantitatively detected by combining a specific nuclease with a RNA specific fluorescent dye. The method specifically comprises the following steps: digesting DNA by specific nuclease to reserve RNA, eliminating interference, and then quantitatively detecting RNA residues by using the RNA specific fluorescent dye; or, after the RNA is digested by the specific nuclease, quantitatively detecting the RNA by using the RNA specific fluorescent dye, and calculating a difference value before and after digestion to quantitatively detect the RNA residue. Compared with conventional AGE qualitative detection, the method provided by the invention is quantitative detection,and because the RNA specific fluorescent dye has high sensitivity to RNA and is theoretically less influenced by the length difference of RNA fragments, the method provided by the invention has the advantages of good quantitative linearity, high sensitivity, small RNA fragmentation interference and good accuracy; compared with a conventional RT-qPCR mode to indirectly quantifies the RNA residues,the method directly quantifies the RNA residues, is simple and rapid in experimental process and low in cost; theoretically, the method directly quantifies the RNA with more accuracy, and has low requirements on experimenters.

Description

technical field [0001] The present invention relates to the field of biology, in particular to a method for detection and quantification, and in particular to a general quick and simple quantitative detection method for RNA residues in DNA products, such as the quantification of RNA residues in plasmid / minicircle DNA (minicircle DNA) products. Background technique [0002] DNA products represented by plasmid / minicircle DNA can be used as DNA vaccine products or one of the main components of products, and can also be used as important raw materials for viral vector or DNA vector gene therapy, and important raw material for viral vector or non-viral vector cell immunotherapy. The RNA residues of these DNA products may affect their biological activities (such as interfering with plasmid transfection and expression efficiency) or cause risks (such as exogenous RNA can activate cellular interferon pathways to trigger immune responses), so it is necessary to establish the RNA resid...

AI Technical Summary

Problems solved by technology

The AGE method can only be used for qualitative detection of RNA residues, but commonly used AGE nucleic acid dyes (such as EB, SYBR Safe, SYBR Green, GelRed, etc.) are far less sensitive to RNA than DNA, and DNA components in samples will seriously affect RNA exposure imaging (like figure 1 In Lane1, when 10ng / µL RNA is normally exposed, the relatively high concentration of DNA is severely overexposed, which interferes with the detection of RNA bands), and the actual residual RNA is RNA degradation fragments rather than complete rRNA, which further reduces the sensitivity of the AGE method. Sensitivity and Accuracy

RT-qPCR method (such as Huzhou Shenke-1201201, E. coli Total RNA Residue Detection Kit) The experimental process is complex, RNA standards with poor stability need to go through complex processing procedures (RNA extraction, reverse transcription, and then qPCR quantification), and DNase digestion conditions need to be optimized for the sample type

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