Abstract
ABSTRACTThe effectiveness and economics of polyvinyl sulfonic acid (PVSA) as a ribonuclease inhibitor for in vitro systems is reported. PVSA was shown to inhibit RNA cleavage in the presence of RNase A as well as in the presence of Escherichia coli lysate, suggesting that PVSA can act as a broader ribonuclease inhibitor. In addition, PVSA was shown to improve the integrity of mRNA transcripts by up to 5-fold in vitro as measured by their translational viability. Improved preservation of mRNA transcripts in the presence of PVSA under common RNA storage conditions is also reported. A cost comparison with commercially available RNAse inhibitors indicates the economic practicality of PVSA which is approximately 1,700 times less expensive than commonly used ribonuclease inhibitors. PVSA can also be separated from RNA by alcohol precipitation for applications that may be sensitive to the presence of PVSA.
Highlights
RNA plays a vital role in myriad biologic processes including protein translation, gene regulation, and gene expression
RNA degradation is a major challenge for in vitro applications such as cell-free protein synthesis (CFPS), reverse transcription polymerase chain reaction (RTPCR), quantitative RT-PCR, RNA-Seq and Northern Blot analysis, all of which rely on RNA integrity and purity.[5,6,7,8]
To examine the RNase inhibitory potency of polyvinyl sulfonic acid (PVSA), we measured the ribonuclease activity of RNase A and E. coli lysate in the presence of PVSA
Summary
RNA plays a vital role in myriad biologic processes including protein translation, gene regulation, and gene expression. One prominent solution is the pretreatment of samples and solutions with diethylpyrocarbonate (DEPC), which is effective for ribonuclease inhibition.[9,10] One issue with this solution, is that DEPC and other similar chemicals are known carcinogens and require caution and training for their use. These chemicals react quite readily with amine, thiol, and alcohol groups and cannot be used in many biologic experiments where buffers and biologic reagents being used and produced often contain these side groups. Produced RNase inhibitors may effectively inhibit ribonucleases, but their action is often specific to certain types of ribonucleases and they are often very expensive.[9,12,13]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
