Checking extraction conditions for 197N and 268N
Date: 10/29/20
Tissues used for this experiment: 197N Colon and 268N Spleen. Almost no RNA was extracted from 197N and total RNA extracted from 268N was degreed.
According to information on Fisher website: https://www.thermofisher.com/us/en/home/references/ambion-tech-support/rna-isolation/tech-notes/isolation-of-total-rna-from-difficult-tissues.html
“Rat spleen and thymus are high in nucleases and nucleic acids. Efficient homogenization is critical for reducing the effects of nucleases found in the tissues. The high DNA and RNA content of these tissues causes the homogenates to be unusually viscous. Extraction of such viscous homogenates sometimes results in incomplete phase separation. Adding more lysis solution and/or re-extracting with phenol:chloroform:IAA will help alleviate this problem. Also, multiple phenol:chloroform:IAA extractions can be performed to ensure the partitioning of DNA into the organic phase during the acid phenol extractions of the RNA isolation procedure."
From previous experiment, we found 197N and 268N cell extracts were very viscous with sonication in buffer with or without EDTA. Here we add sonication step (different pulse time) to see whether sonication can reduce the viscosity and produce a large volume of clarified lysate.
Weigh out 50mg of powder and add 200ul (1:4) or 450ul (1:9) of extraction buffer (EB), use the old sonicator at setting 3, sonicate for 1, 3 or 5 sec
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Transfer the supernatant to fresh tubes for protein assay and gel analysis; take a picture of the pellet left in each tube (from left to right samples 1-10)
It looks like that adding EDTA to the extraction buffer will make the cell lysate more viscous in some tissues. Sonication can help reduce the viscosity. Increase the volume of extraction buffer may further increase the extraction efficiency. (*After centrifugation step to get clarified lysate, this sample seemed to have less volume that other tubes which indicated the extraction buffer added might be less than 200ul to start with).
Load 0.5ul or 1ul of cell extract on gel (see Table above)
Next steps:
For 268N (spleen): add more extraction buffer (1:9 w:v) and add sonication 5 sec For 197T and N (colon): try sonication; also try 162T and N (colon)