RNA isolation from Oocytes - (Oct/01/2006 )
This is my first my first post, but I am in need of some assistance of purifying RNA from bovine oocytes and early embryos (anything with a zona). It has been a problem in our lab for about 6 months. I am a new graduate student 2 months ago and this has been passed onto me.
Currently, we are using Ambion's mirVana kit. We are getting some results from high numbers of hatched blastocysts (50) and cumulus only purifications but would like to reduce the number to 10-20 oocytes-->Blastocyst. We have tried removing the zona in 3-4 min of .5% pronase; heating in lysis buffer 90 C x 5 min, followed with vortexing 2 min; physically cutting the zona with a laser, eluting with 50 ul instead of the recommend 100 ul and all resulted in negative results to produce cDNA. Also, I tried treating with DNAse and running the RT-PCR in the same tube instead of using the spin columns.
Any help or direction to find a succesful protocol would be greatly appreciated!
I've never purified RNA from bovine oocytes or embryos, but I frequently purify RNA from as little as 10 mouse oocytes.
We didn't get good results using kits either, so we simply use TRIZOL. If the zona is a problem I am sure TRIZOL can remove it. We use a standard procedure for TRIZOL, but always add 1 ug glycogen as a carrier (otherwise you'll never see the pellet). When using 10-20 oocytes I resuspend the pellet in a small volume and use the entire amount for RT. Let me know if you want the exact protocol.
Thank you for your helpful suggestion! If you would not mind sharing the protocol so I could compare some quantities and times that would be greatly appreciated.
Thank you and I hope you have a Great week!
Best of Luck,
Harvest oocytes and transfer to a clean drop of media. Then pick up oocytes with a small volume of media (usually 10-20 ul) and transfer to a tube containing 1 ml TRIZOL. You can store the oocytes in TRIZOL at -20 until you're ready to purify the RNA (I always do this, so it's possible that freezing helps the oocytes to dissolve).
~Remove from -20 and leave at RT until melted.
~Add 0.2 ml chloroform, shake by hand 15 sec.
~Incubate 2-3 min at RT.
~Spin at 12,000 rpm, 15 min, 4 degrees C.
~Transfer the colorless upper phase to a clean tube (~0.6 ml).
~Add 0.5 ml isopropyl alcohol and 20 ug glycogen (that was a mistake above, I add 1 ul of glycogen from Invitrogen which is 20 ug/ul).
~Mix well by hand and incubate at -20 at least overnight (this is very important to get a good RNA yield).
~Spin at 12,000 rpm 10 min, 4 degrees C.
~Discard supernatant slowly and completely using a pipetman, RNA forms a gel-like pellet.
~Wash RNA pellet with 1 ml of 75% EtOH (in DEPC H20).
~Spin 9500 rpm 5 min, 4 degrees C.
~Discard EtOH carefully, using a pipetman.
~Short spin to collect remaining EtOH at the bottom.
~Discard EtOH carefully and completely by using a fine tip pipetman.
~Air dry 5 min (do not let the pellet dry out completely).
~Dissolve RNA pellet in DEPC H20. For less than 20 oocytes, I dissolve in the volume I want to add to the RT PCR reaction + 0.5 ul.
~Incubate RNA at 55-60 degrees C for 10 min to dissolve completely.
~Use immedietly for RT or store at -20.
Note, the amount of RNA is too small to quantitate by spec, so I just quantitate by the number of oocytes I used. I get very consistent results for L19 this way.
Thank you Zona, it worked GREAT!!!!!!! Finally a little glimmer of success for the lab, now its time to really work..... doh.
Thanks again and good luck