In a Qiagen RNA isolation kit either DTT or BME has to be added to the RLT buffer.
With respect to proteins, usually beta-mercaptethanol (BME) forms (irreversible) covalent bonds with cysteine (sulfur containing) amino acid side chains which prevents oxidation (formation of intra/inter protein bonds) and dithiothreitol (DTT) also prevents oxidation but via a redox reaction with itself and is benign with respect to protein folding/structure.... Whatever, the point is that RNAses which will stuff up your RNA extraction can be deactivated with these chemicals during RNA extraction, BME is a sure bet so I would go with that - prep in a fume hood/tissue culture hood, it pongs worse than DTT, people get annoyed with it these days so just make sure your disposal routes are contained, clean up any spills immediately - bleach can oxidise it effectively if you do have a spill...
With respect to proteins, usually beta-mercaptethanol (BME) forms (irreversible) covalent bonds with cysteine (sulfur containing) amino acid side chains which prevents oxidation (formation of intra/inter protein bonds) and dithiothreitol (DTT) also prevents oxidation but via a redox reaction with itself and is benign with respect to protein folding/structure.... Whatever, the point is that RNAses which will stuff up your RNA extraction can be deactivated with these chemicals during RNA extraction, BME is a sure bet so I would go with that - prep in a fume hood/tissue culture hood, it pongs worse than DTT, people get annoyed with it these days so just make sure your disposal routes are contained, clean up any spills immediately - bleach can oxidise it effectively if you do have a spill...
Article Replacing β-mercaptoethanol in RNA extractions
I found this article to be informative.
"Overall, we found DTT to be more effective than β-ME in deactivating RNases but that using β-ME is better than using no reducing agent. The beneficial effect of using β-ME rather than no reducing agent was previously noted when Qiagen’s automated QIAsymphony technology was used to extract RNA from white blood cells that was subsequently analyzed by qRT–PCR [5]. Our results concur but also show that DTT could have been used instead, probably more effectively. Not only can DTT be used at a lower concentration than β-ME, but it is also less toxic and pungent; oral LD50 values (rat) are 244 mg/kg for β-ME but 400 mg/kg for DTT, and environmental LC50 values (Daphnia magna in 48 h) are 1.52 mg/L for β-ME and 27 mg/L for DTT [8], [9]. DTT has disadvantages compared with β-ME in that it has a shorter half-life and is supplied as a powder rather than as a solution, so it must be either freshly made up before sample homogenization or stored as frozen aliquots. However, we believe that the functional, practical, and safety-related advantages that DTT offers compared with β-ME clearly outweigh its more complex preparation and storage requirements. We believe that the widespread use of β-ME in RNA extractions can now justifiably be questioned, and we urge manufacturers of spin column purification kits to actively promote the less toxic DTT alternative."
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques