Hi,

I would really appreciate your help or advice. I have struggled to get good quality RNA from colon biopsies - RIN averaging 2 on the Tapestation. I have tried many methods / variations with no luck:

1) Biopsies that I have collected from colonoscopy are immediately (within 1 minute) placed in:

a) autoclaved PCR eppendorphs that contain 1ml of RNAlater (from Sigma) - which I have either left at room temperature for 24 hrs before freezing it at -80, or immediately placed in liquid nitrogen then transferred to -80 or processed immediately. 

b) cryovial and immediately placed in liquid nitrogen. 

c) placed in RLT buffer and placed in liquid nitrogen I have tried

2) Different kits:

a) Powermicrobiome RNA kit by Mobio

b) RNeasy Minikit

3) Different RNA extraction benches and RNAse free waters for elution!

4) Processed the same day or 24 hours later - thawed on ice if -80C

5) Extraction done on ice and at room temperature

As soon as I extract the RNA, I place it on ice and either quantify it and run it on the tapestation the same day or place it in a -80 freezer till day of analysis (thawed on ice). I am not sure where I am going wrong and I would be grateful if you could tell me if there is a protocol that works or if I am missing a trick!

Thanks.