A general and rapid method for the quantification of rna residues in dna products

Abstract

The invention discloses a method for quantifying RNA residues in general and rapid DNA products. The method uses specific nucleases combined with RNA-specific fluorescent dyes to quantitatively detect RNA. The method specifically includes: specific nuclease digests DNA to retain RNA, eliminates interference, and then uses RNA-specific fluorescent dyes to quantitatively detect RNA residues; or, after specific nuclease digests RNA, uses RNA-specific fluorescent dyes to quantitatively detect RNA, and calculates the digest The difference before and after was used to quantitatively detect RNA residues. Compared with conventional qualitative detection of AGE, the method of the present invention is a quantitative detection. Since RNA-specific fluorescent dyes have high sensitivity to RNA and are theoretically less affected by differences in the length of RNA fragments, the present invention has good quantitative linearity and high sensitivity, and is not affected by RNA fragments. Small chemical interference and good accuracy; compared with conventional RT-qPCR indirect RNA residue quantification, this method directly quantifies RNA residues, the experimental process is simple, fast, and low cost. Theoretically, direct RNA quantification is more accurate, and requires Low.

Description

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

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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