How to achieve RNA extraction of nucleus and cytoplasm in SH-SY5Y cells?

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Skylar King

I have used NE-PER Nuclear and Cytoplasmic Extraction reagents from Fisher Scientific and they work well. Cat#78833. I also found that passing the nuclear pellet through a 27g needle several times helps break the nuclear membrane so you can extract the nuclear proteins more efficiently. Draw up slowly and rapidly eject 5-10 times. Hope this helps!

Aarya Chitransh

It's commendable that you're trying to optimize your protocols for the separation of nucleus and cytoplasm in different cell types. Since you've had success with HeLa cells but are encountering sensitivity issues with SH-SY5Y cells, here are some suggestions to consider:

Optimize buffer conditions: The composition of RLN buffer can affect the efficiency of nucleus-cytoplasm separation. You may need to adjust the concentration of NP40 or other components in the buffer to better suit the sensitivity of SH-SY5Y cells. Try varying the NP40 concentration (within a range that maintains cell integrity) or consider using alternative lysis buffers optimized for neuronal cells.

Gentle cell lysis: SH-SY5Y cells may be more sensitive to harsh lysis conditions. Ensure that you're performing gentle cell lysis to minimize cellular damage. You can try reducing the duration or intensity of cell lysis or exploring alternative lysis methods that are less disruptive to neuronal cells.

Optimize cell harvesting: Ensure that you're harvesting SH-SY5Y cells under conditions that preserve cell integrity. Avoid over-trituration or excessive mechanical disruption during cell harvesting, as this can lead to increased sensitivity to lysis conditions.

Assess cell viability: Before and after cell lysis, assess the viability of SH-SY5Y cells using appropriate viability assays (e.g., Trypan blue exclusion, MTT assay). This can help determine whether the sensitivity issues are due to cell death induced by the lysis procedure.

Consider alternative methods: If you're still facing challenges, consider alternative methods for nucleus-cytoplasm separation in SH-SY5Y cells. Techniques such as differential centrifugation, gradient centrifugation, or commercially available kits optimized for neuronal cells may provide better results.

Consult literature: Look for published protocols or studies that have successfully performed nucleus-cytoplasm separation in SH-SY5Y cells. These resources can provide valuable insights and guidance for optimizing your protocol.

By systematically optimizing your protocol and considering the specific characteristics of SH-SY5Y cells, you can improve the efficiency and sensitivity of nucleus-cytoplasm separation for downstream applications.