I am looking for a proper protocol for RNA extraction from breast cancer tissue. I conducted a number of trials and was unsuccessful. I got RNA quantity, concentration = 100ng/ul 260/280= 1.56,1.68.1.78, 1.82, 1.90, 1.98 and 260/230= .68, 0.079, 1.00, 1.34 etc., but when I checked the RNA quality in denaturing MOPS gel, there were no signs of the RNA subunit bands.
I am using tissue frozen in RNALater and keep at -80 until use. I tried pestle and mortar homogenization. I have done the both teh protocol of Trizol method and of the ambion kit but the result was the same.
If the tissue is fresh, I go with the RNAeasy method, which is a modified Boom et al., extraction (I can find the paper if you really want). If the tissue is frozen, I work with that same Qiagen kit. If the tissue is Formalin Fixed Paraffin Embedded (FFPE), I use a kit from Omega bio-tek which is based on a silicated magnetic bead patent authored at MIT-Whitehead some years ago. The name of the Omega bio-tek kit is E.Z.N.A. Mag-Bind FFPE RNA Kit. One thing though about FFPE is that it will not give you nice ODs and the RNA is somewhat degraded, so I wouldn't use it for Northerns, for instance. However, it is fine for qPCR, nanostring, Affy chips and many other applications (RNAseq). Good luck.
The breast cancer cell has too much lipids. Therefore I recommend to use RNeasy lipid tissue kit for isolation of RNA (Qiagen). hey also provide the protocol.
For frozen tissue it would be better while homogenisation keep the temperature low. So, continuously add liquid nitrogen to maintain the RNA.
hi, I am encountering a similar situation, I tried to extract RNA from frozen breast cancer tissue, when I added Trizol I felt that the quantity of RNA will be abundant(100mg tissue or more and the solution felt sticky), however when I added isopropanol and precipitate at 4℃(trying -70℃ overnight now but I don't know if it will work) then centrifuge, there is barely any RNA pellet visible, the upper phase of chloroform was a bit yellow, I am not sure if its the lipids in the tissue at work here.
breast cancer tissue was resected then kept in liquid nitrogen until use, I am using the protocol to extract RNA from cultured cells, just used a tissue homogenizer to crumble the tissues before I add chloroform.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA