Hello David.

Frequent failures of RNA isolation of any single cell suspension or tissues from any sources may be caused by improper sterile technique, incomplete breakage, and presence of RNases released from the tissues, contamination from the surrounding including the experimenter during cell lysis process.

- Wash your cells in DEPC-treated PBS three times, and make hard cell pellets in aliquoted E-tubes at 5,000rpm for 10 min in the last wash.

- Snap freeze the E-tubes by submerging into LN2 gas.

-Transfer the cell pellets via sterile hypodermic needle into a DEPC-treated, pestle and mortar that are previously stored at -80C overnight.

- Grind several cell pellets together while pouring some LN2 gas over them to make cell powder finely.

- Then follow the normal total RNA isolation procedure accordingly.

You never fail.

Best wishes.

Dear Sang Ho Lee,

Many thanks for your suggestion. I have to consider it as an option.

Best wishes,

David

It seems that mechanical pre-homogenisation with 21G needles followed by QIAshredder homogenization with the addition of RLT lysis buffer and 2-mercaptoethanol provide adequate RNA concentration after extraction with RNeasy Mini Kit.

Hi David,

Was this work with Myxobolus cerebralis? I am curious what amount of RNA per triactinomyxon you yielded.

Thank you,

Ben