TITLE: Collection and shipment of live skeletal muscle for RNA and cell isolation
AUTHORS: Prech Uapinyoying

[DESCRIPTION]
Collecting and transporting fresh muscle tissue from a clinical site for experimentation at a remote location can be a logistical challenge. This protocol provides the procedures for collecting, preserving and shipping fresh skeletal muscle autopsy samples from the morgue to a remote laboratory site for downstream experiments requiring viable cells and
high-quality RNA such as single-cell RNA-seq.



[GUIDELINES]
Tissue collection for this protocol needs prior approval by the users' Institutional Ethics Board or equivalent ethics committee.

[STEPS]
SECTION: Sample collection &amp; preparations
1. At sample collection site, communicate and schedule with mortician to come pick up the muscle autopsy sample, ensuring samples be collected 48 hours or less postmortem.
 

 

 

1.1. Record relevant details about the sample such as: the site name, subject ID, age of donor when sample collected, sex assigned at birth, gender, ethnicity, cause of death, medications and medical history, BMI, postmortem time, muscle group, and collection date. These details may be relevant to downstream experiment.
2. Collect one gram (1000 mg) or more of quadricep muscle sample such that it does not dry during the procedure*.

3. Place the harvested muscle into a 50 mL conical tube half filled with chilled transplant solution on ice . Place the tube in a 1.2-gallon (or larger) polyurethane or insulated foam bucket full of wet ice. Close the lid and quickly transport to the lab.
 

 



4. In the laboratory and prepare to weigh the muscle sample by placing a small or medium empty weigh boat on a laboratory scale, and tare.
5. Working quickly, remove the wet muscle using a pair of forceps from the tube and absorb excess transplant solution from the muscle tissue by wicking on clean Kim wipes. Further, remove the excess liquid by thoroughly dabbing the tissue on a fresh Kim Wipe.
6. Immediately transfer the muscle onto the weigh boat, record the weight and moisten the muscle by placing it into the original tube of chilled transplant solution.
7. Take the wet muscle piece back out of the transplant solution and place on a medium weigh boat.
8. Using a clean razor blade, roughly divide the muscle into pieces smaller than 250 mg and place each piece into an individual cryotube.
 

 


SECTION: Preserving and shipping samples
9. Prepare 500 µL of preservation buffer by mixing 497.5 µL of chilled transplant solution and 2.5 µL of RNAse inhibitor (40 U/mL stock) for a final concentration of 0.2 U/mLper piece of muscle into individual cryotube.   

SECTION: Preserving and shipping samples
10. Submerge each of the divided muscle pieces into individual cryotube with preservation buffer. Close the screw caps, label with sample ID and date, and place on wet ice.   
SECTION: Preserving and shipping samples
11. Prepare for shipping by placing 1-2 freezer packs into an insulated foam box fitted inside a cardboard shipping box and add wet ice on top. Place the sample tubes into the wet ice.
SECTION: Preserving and shipping samples
12. Close the foam and cardboard boxes and tape the seams shut using packaging tape. Place label and send the package for overnight shipping.