wht is the best way to extract RNA from oocytes stages - (Mar/02/2007 )
Can any one please tell me, whts the best way to extract RNA from different stages of oocytes using Trireagent??
should I use Isopropenol combind with high salt at precipitation step??
Am I supposed to keep it in -20 overnight after adding isopropenol in it??
which one is better to use chloroform or BSP??
Thx in advance.
Here's my protocol, which works great for me:
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.