Zeynep ZÇ Çeli̇k The pellet may not dissolve during manual RNA isolation from MCF-7 spheroids due to incomplete lysis of the dense 3D structure, which contains tight cell-cell junctions and extracellular matrix components. Inadequate homogenization or insufficient incubation with lysis buffer (e.g., TRIzol) can leave intact spheroid cores, leading to RNA entrapment or contamination with proteins or genomic DNA. To resolve this, ensure thorough mechanical disruption (e.g., pipetting or needle shearing) and complete dissolution in lysis buffer before proceeding to phase separation.

Hi Rajarethinam, thank you so much for your reply. It was enlightening for me.

The failure of the pellet to dissolve during manual RNA isolation from MCF-7 spheroids is a common issue, particularly when dealing with 3D cell models like spheroids, which are more compact and rich in extracellular matrix components compared to 2D cultures. Several factors can contribute to this problem:

• Incomplete Lysis or Homogenization: Spheroids are dense clusters of cells with strong cell-cell junctions and can be particularly resistant to lysis. Insufficient homogenization or too little lysis reagent can leave behind undissolved material, resulting in a persistent pellet.
• High Content of Extracellular Matrix, Polysaccharides, or Proteins: The insoluble pellet may contain extracellular matrix proteins, polysaccharides, or other biopolymers that do not dissolve easily in typical RNA extraction reagents. This is also seen in tissues or samples with high carbohydrate or protein content.
• Overdrying the Pellet: If the RNA pellet is overdried after precipitation, it can become very difficult to redissolve.
• Improper Solubilization Conditions: Not resuspending the pellet in the correct buffer or failing to use adequate heat or agitation can make dissolution difficult. Some protocols suggest gentle and repeated pipetting in DEPC-treated water or SDS solution, followed by incubation at 55–60°C to help redissolve the pellet.
• Excessive Centrifugation Speeds: Centrifuging at speeds higher than recommended can compact the pellet tightly, limiting solubility.
• Carryover of Starch or Contaminants: In some cases, especially with plant or certain cell types, undissolved material may be starch, glycogen, or similar substances that typical RNA extraction methods do not remove.

To resolve this, ensure complete lysis of the spheroids, consider mechanical disruption (such as bead-beating or vigorous pipetting), increase the amount of lysis buffer, avoid overdrying, and try to resuspend the pellet by warming and gentle mixing. If the problem persists, adopting protocols optimized for 3D cultures or samples with high extracellular matrix content may be necessary.