In vitro application of ribonucleases: comparison of the effects on mRNA and miRNA stability

BMC Research Notes

Bernd Denecke¹  Arian Aryani¹ 

[1] Interdisciplinary Center for Clinical Research Aachen (IZKF Aachen), RWTH Aachen University, Pauwelsstrasse 30, Aachen, Germany

关键词: RNA integrity;    RNA stability;    Ribonuclease;    Ribosomal RNA;    miRNA;    mRNA;   

Others  :  1177872
DOI  :  10.1186/s13104-015-1114-z

 received in 2013-12-11, accepted in 2015-03-31,  发布年份 2015

【 摘 要 】

Background

MicroRNA has become important in a wide range of research interests. Due to the increasing number of known microRNAs, these molecules are likely to be increasingly seen as a new class of biomarkers. This is driven by the fact that microRNAs are relatively stable when circulating in the plasma. Despite extensive analysis of mechanisms involved in microRNA processing, relatively little is known about the in vitro decay of microRNAs under defined conditions or about the relative stabilities of mRNAs and microRNAs.

Methods

In this in vitro study, equal amounts of total RNA of identical RNA pools were treated with different ribonucleases under defined conditions. Degradation of total RNA was assessed using microfluidic analysis mainly based on ribosomal RNA. To evaluate the influence of the specific RNases on the different classes of RNA (ribosomal RNA, mRNA, miRNA) ribosomal RNA as well as a pattern of specific mRNAs and miRNAs was quantified using RT-qPCR assays. By comparison to the untreated control sample the ribonuclease-specific degradation grade depending on the RNA class was determined.

Results

In the present in vitro study we have investigated the stabilities of mRNA and microRNA with respect to the influence of ribonucleases used in laboratory practice. Total RNA was treated with specific ribonucleases and the decay of different kinds of RNA was analysed by RT-qPCR and miniaturized gel electrophoresis. In addition, we have examined whether the integrity observed for ribosomal RNA is applicable to microRNA and mRNA. Depending on the kind of ribonuclease used, our results demonstrated a higher stability of microRNA relative to mRNA and a limitation of the relevance of ribosomal RNA integrity to the integrity of other RNA groups.

Conclusion

Our results suggest that the degradation status of ribosomal RNA is not always applicable to mRNA and microRNA. In fact, the stabilities of these RNA classes to exposure to ribonucleases are independent from each other, with microRNA being more stable than mRNA. The relative stability of microRNAs supports their potential and further development as biomarkers in a range of applications.

【 授权许可】

   
2015 Aryani and Denecke; licensee BioMed Central.

【 图 表 】

Figure 1.

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