Hy, in my research I need to extract RNA from mice gut samples for further cDNA and qPCR. However, the results are really inconsistent, sometimes it's 700ng/µl, and for another replicate which has the same sample we get 350ng/µl, surprisingly it always seems that the first sample is the best and afterwards the quantity drops dramatically. We have found that it is really dificult to digest the samples since the gut samples are quite rubbery. The methods we are currently using is cutting the tissue in small pieces, adding Qiazol, trying to stamp the tissue with a micro mortar and vortex afterwards with a vortex for 30-45 min (in between vortexing we cool the samples on ice for 5 min). After phase separation with chloroform we are really carefull to only extract aqueous phase and premix it with 100% ethanol. Then we use the miRNeasy Kit for RNA isolation.
We can't think of any other reason for the inconsistency to be lying with the digestion method in the way we stamp and cut the tissue.
Also the other issue is that we always have low A260/230 ratios but really good A260/280 ratios. Perhaps this is due guanidine contamination.
Can anyone give some insight in what can be changed? Ofcoarse a tool such as a tissue ruptor with be handy, but we don't have acces to it at the moment, perhaps we can try liquid nitrogen, but this would be really time consuming when using a mortar and pestle (I need to do 85 samples).
Here's a summary of strategies to improve RNA yield and purity from mouse gut samples:
1. **Optimizing Homogenization**:
- Use a **bead homogenizer** (if available) or **freeze samples in liquid nitrogen** and grind them with a mortar and pestle for better cell lysis. Alternatively, mince tissues finely with scissors to make them easier to lyse.
2. **Adjust RNA Extraction Protocol**:
- Extend vortexing time with QIAzol to at least 45 minutes, and consider pre-incubating the sample with QIAzol for 5–10 minutes.
- Perform 1–2 freeze-thaw cycles after initial QIAzol treatment for more effective tissue breakdown.
3. **Improve A260/230 Ratios**:
- Add an **extra 80% ethanol wash** step to reduce contaminants.
- Slightly increase chloroform during phase separation (0.25–0.3 mL per 1 mL QIAzol) for purer RNA separation.
4. **Consistency in Processing**:
- Process samples at a uniform time to avoid yield drops due to time variations.
5. **Additional Cleanup for Guanidine Contamination**:
- Increase ethanol during binding or add a post-extraction cleanup (e.g., using a lithium chloride precipitation) to remove salts.
These steps should help improve RNA consistency and purity without extra equipment.
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