Hi, Angélique Buton

I'm not sure if you've considered including a pre-incubation step with enzymes; perhaps extracellular matrix components or collagen might be interfering. In my case, when working with highly fibrotic tracheal tissues, we faced similar issues with low protein yield after tissue dissociation. Like you, we initially tried mechanical, dissociation using liquid nitrogen, but the protein concentrations we obtained were insufficient. However, after pre-incubating the samples with enzymes such as collagenase, hyaluronidase, and trypsin, we achieved much more effective tissue dissociation.

After that, we performed ultrasonic homogenization on ice and extracted proteins using DTT, acetone, and trichloroacetic acid. The protocol might need some adjustments in your case, especially since you're planning to extract RNA, which is an important factor to consider.

Nonetheless, for applications like Western blot, ELISA, or even flow cytometry, this approach could work quite well.

¡Best of luck!

It's tricky to get good quality RNA and proteins using just one protocol. Most folks would homogenize & then split the samples to use part for protein and part for RNA.

For the RNA, have you tried using proteinase K? That helps break down tougher tissues.

I developed methods to extract good quality RNA (sorry not protein) from fish bones by: 1) preserving the bones / skin in RNA later, then 2) disrupting the tissues with liquid nitrogen, 3) adding an appropriate amount of trizol then 4) transferring the disrupted bone tissue / trizol mix to tissue homogenisation tubes and running them through a bead beater. The RNA could then be extracted using a combination of trizol / Qiagen RNeasy extraction methods: Article Shaping development through mechanical strain: The transcrip...

One important point to note, was that I got the best RNA quality and yields by optimising the loading of the tissue homogenate onto the extraction column. I tried loading a dilution series of homogenate onto the columns to identify the amount that produced the highest quality RNA. The optimal loading conditions reduced the amount of contaminants that were washed through the column. The optimal loading concentration is actually quite a bit less than what Qiagen recommends, potentially due to the high amounts of collagen and fat in the bone.

This method of optimising extraction column loading was used to develop RNA extraction protocols from salamander skin, so I think it's pretty transferrable to different "difficult" tissue types. Article Functional colour genes and signals of selection in colour‐p...

I suggest you use 2 separate protocols for each as said by Katie. Stabilise RNA as suggested by Helen with RNALater/Shield/Hold . Depending on what you are aiming to analyse the protein for use enzymes proteinase K (if using Trizol you can get it from the layers) or protein specific enzymes.

Hello!

Mouse skin is tough and fibrous, making homogenization for protein and RNA extraction particularly challenging. To improve your yields, first finely mince the tissue on ice and consider a brief enzymatic digestion with low-concentration collagenase or dispase to soften the extracellular matrix without damaging intracellular contents. For RNA extraction, strong lysis buffers like Trizol or QIAzol are essential, while for proteins, use RIPA buffer with protease and phosphatase inhibitors or a stronger SDS-based buffer if needed. Whether using a bead mill homogenizer (eg. the bullet blender from Next Advance https://www.nextadvance.com/bullet-blender-homogenizer/ ) or liquid nitrogen with a mortar and pestle, ensure the tissue is processed into a fine powder and work quickly on ice to preserve integrity. During bead homogenization, use ceramic or steel beads and perform short, high-speed bursts with cooling intervals to avoid overheating. After homogenization, centrifuge at high speed to clarify the lysate. Always minimize handling time, keep samples ice-cold, and use low-binding tubes to prevent sample loss.