I did the RNA extraction two times (first time in our lab and second time by a company lab) from rabbit scleral tissue and the degradation was very severe. The RIN values were very low in all samlples and we don't know the reason behind it. Please if there is any one could help me with this I will be so thankful.
I understand your frustration! RNA degradation during extraction from rabbit scleral tissue can be a real challenge. Here are some possible reasons and solutions you can consider:
Possible reasons for RNA degradation:
- RNase contamination: RNases are enzymes that break down RNA. Even small amounts of RNase contamination can significantly degrade your RNA. Make sure your lab is following best practices for RNase-free techniques, including using nuclease-free water and wiping down surfaces with RNase decontamination solutions.
- Tissue handling: Scleral tissue might require specific handling procedures to minimize RNA degradation. This could include snap-freezing the tissue immediately after collection and storing it at -80°C until extraction. You might also need to adjust homogenization techniques to minimize RNA shearing.
- Extraction method: Not all RNA extraction methods are created equal. Some methods might be better suited for tough tissues like sclera. Consider using a commercially available kit specifically designed for fibrous tissues or low-abundance RNA.
Here are some next steps:
- Compare protocols: If the RNA degradation occurred in your lab, compare your extraction protocol to the company protocol used for the second extraction. See if there are any key differences in handling or extraction methods.
- Optimize extraction time: RNA degradation can occur over time. Try optimizing your extraction workflow to minimize the time between tissue collection and RNA isolation.
- Run a quality control step: Before proceeding with downstream applications, run a quality control step on your RNA using a microfluidic analysis platform or gel electrophoresis. This will help you assess the severity of the degradation and determine if the RNA is usable for your experiments.
If you've tried these suggestions and are still having trouble, consider reaching out to the company that performed the second extraction. They might be able to provide some insights into their protocol or troubleshoot potential issues specific to scleral tissue.
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