Hello!
I am using the AccuPrep® Universal RNA Extraction Kit(https://us.bioneer.com/pagecat1/accuprep/universal-rna-extraction-kit?tab=overview), which requires adding 10 µL of 99% β-mercaptoethanol (BME) per 1 mL of RB Buffer. Since I used 850 µL of RB Buffer, I added 8.5 µL of 99% BME inside a fume hood and proceeded with the RNA extraction process.
Since this kit follows a spin column-based protocol, I needed to discard the flow-through during centrifugation. However, I made the mistake of performing the extraction process on a clean bench instead of inside the fume hood. At this stage, the BME odor became extremely strong, causing me to feel a sudden wave of heat in my body and mild dizziness. While I don’t think it was a serious reaction, I am concerned about long-term exposure risks because my lab has poor ventilation, and it is impractical to perform the entire RNA extraction process inside a fume hood.
To address this issue, I am considering using DTT as a substitute for BME as DTT is also a strong reducing agent and is expected to have a similar function in inhibiting RNase activity while being less volatile and odorless.
I would like to know:
• Would DTT provide equivalent reducing conditions in this protocol?
• What would be the appropriate concentration of DTT if used instead of BME?
• Are there any potential drawbacks to using DTT in this protocol?
• Since DTT has low volatility, can I safely handle it outside of a fume hood?
I would greatly appreciate any insights or recommendations. Thank you!
you can deploy copilot It's definitely important to address safety concerns in the lab. Let's go through your questions one by one:
### Would DTT provide equivalent reducing conditions in this protocol?
Yes, **dithiothreitol (DTT)** can provide equivalent reducing conditions in RNA extraction protocols. DTT is a strong reducing agent like BME and can effectively inhibit RNase activity.
### What would be the appropriate concentration of DTT if used instead of BME?
The appropriate concentration of DTT is typically **20 µL of 2M DTT per 1 mL of buffer** be used instead of BME in Buffer RLT ... - QIAGEN](https://www.qiagen.com/us/resources/faq?id=23023950-623b-42e2-94f4-f89c28101ce5&lang=en). This should be equivalent to the concentration of BME used in your protocol be used instead of BME in Buffer RLT ... - QIAGEN](https://www.qiagen.com/us/resources/faq?id=23023950-623b-42e2-94f4-f89c28101ce5&lang=en).
### Are there any potential drawbacks to using DTT in this protocol?
While DTT is less volatile and less odorous than BME, it can still pose some risks. DTT can be a skin irritant and should be handled with care. Additionally, DTT might not be as effective as BME in some specific protocols, so it's always good to check the literature or manufacturer's recommendations for your specific kit.
### Since DTT has low volatility, can I safely handle it outside of a fume hood?
Yes, DTT has low volatility and is less hazardous in terms of inhalation compared to BME. However, it's still a good practice to handle it with care and use personal protective equipment (PPE) such as gloves and lab coat. If possible, using a fume hood is still recommended for added safety.
I hope this helps! Good luck
Yes, You can use,
DTT(dithiothreitol), instead of B- Mercaptoethanol in RNA extraction lysis of buffer.
DTT is less toxic and pungent than β-Mercaptoethanol,.
Lower concentration of DTT is useful as compare to β-Mercaptoethanol, but remember that DTT has shorter half life than β-Mercaptoethanol.
DTT always used in this concentration i.e. 20ul of 2M DTT per 1mL of Buffer RLT.
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