Hi, I have tried both. Using Trizol first then a clean up kit, but I found that it is better to just use the Trizol method. However, I have not used frozen cells to do this. I usually collect them directly in 1mL of Trizol. Are you able to grow the cells fresh? I also use this protocol from a former colleague of mine, and it works very well! Hope this helps.

To increase RNA yield and improve the 260/230 ratio when using the Qiagen RNeasy Mini Kit, consider the following strategies: 1. **Additional Wash Step**: Adding an extra wash step before elution can help in removing contaminants that might be affecting the 260/230 ratio. This can be particularly useful if the initial ratio is low . 2. **RNA Cleanup**: After elution, performing an RNA cleanup step can further purify the RNA, potentially improving the 260/230 ratio. This step can help remove any remaining contaminants that might be skewing the ratio . 3. **Combining TRIzol and RNeasy Mini Kit**: Starting with TRIzol extraction followed by cleanup using the RNeasy Mini Kit can be an effective method. TRIzol is known for its ability to efficiently extract RNA, and the RNeasy Mini Kit can then be used to clean up the RNA, reducing contaminants and potentially improving the 260/230 ratio . 4. **Optimizing Sample Preparation**: Ensure that samples are prepared properly, as poor sample preparation can lead to low RNA yields and poor purity. This includes using the correct amount of sample and reagents, and following the protocol closely . 5. **Quality Control**: Regularly assess the quality of your RNA using a spectrophotometer to monitor the 260/230 and 260/280 ratios. This can help in identifying issues early and adjusting the protocol accordingly . 6. **Adjusting Elution Conditions**: Sometimes, adjusting the elution conditions, such as the volume of elution buffer used, can help in improving the purity of the RNA. Using a smaller volume of elution buffer can sometimes concentrate the RNA and reduce contaminants . By implementing these strategies, you can potentially increase RNA yield and improve the 260/230 ratio, which is crucial for downstream applications requiring high-quality RNA.

You will get higher yields (and better quality) if you do an ethanol precipitation after the Trizol prep instead of using the Qiagen columns. But you should still get high enough yields with the Qiagen columns, so the problem is likely elsewhere. Since your cells are frozen, are you sure they're getting completely lysed by the Trizol? If you're working with a frozen cell pellet then you should homogenize the pellet first with something like a bead mill.

Hi I have the same problem. 260/280 is good, but 260/230 is low. The kit is really hard to use. Have you solved your problem now? I am waiting to solve that problem.

Hi,

Thank you all for your helpful responses! I appreciate your insights and suggestions regarding RNA yield and the low 260/230 ratio.

To address this issue, I decided to change the extraction method. I switched from the Qiagen kit to the Zymo Direct-zol kit, which is compatible with Trizol-based extraction. However, I still perform the phase separation step by adding chloroform. As a result, my yield has increased, and the 260/230 ratio has improved.

Great! So happy to hear!

Hi Ana, I have the same problem. Did you figure it out?

Hi Xueyi Cao

To address this issue, I decided to change the extraction method. I switched from the Qiagen kit to the Zymo Direct-zol kit, which is compatible with Trizol-based extraction. However, I still perform the phase separation step by adding chloroform. As a result, my yield has increased, and the 260/230 ratio has improved.