problem with extraction mRNA from oocytos in RNAlater - (Feb/22/2007 )
Hi..
I have a problem.. I tried to extract mRNA using Chomczynski method from oocytos in RNAlater.. I added 1 ml of trizol and after shaking and rotating I still could see two phases - It looked like trizol was one phase and RNAlater was a second phase..I continued extraction but I couldn't see any pellet.. I did PCR reaction using One-Step Kit from Qiagen but I didn't see any product..;( (positive control works well..)
I don't have experience with RNAlater. Mostly I extract mRNA directly from human cells seeded on the plate and It works well..
I supposed that something was wrong and I'm still keeping all the supernatants in -80C so I can repeat extraction but I don't know where I did mistake - I don't want to make the same mistake..
please, give me advice what to do.. ( trully I beg for a fast help..)
Aga
I don't know the composition bout RNA later, but it may contains sthg disturbing for RNAexttaction.
When adding trizol, you normally should see only one phase.
In case of oocytes (and i'm not familiar with them) i guess that if you've removed rna later and if you see 2phase, the reason for 2 phaases may be that youdidn't add enough trizol regarding oocytes volumes. what is the ratio oocytes volume / trizol added?
Hi:
Rnalater should be use only for storage when use the sample it must be wash before making the protocol. You could use PBS 1X and centrifugation to get rid of rnalater. I can't help with the trizol part, because I had never used it, but try washing the oocytes (or any cell) before the nucleotide extraction.
I've never used RNA later, but based on merlav's comment about needing to wash it out before using trizol, I would be vary wary of using it with oocytes. I've found that small numbers of oocytes do not pellet well, so you may lose a lot during the washing step. Or maybe you are simply not able to detect the pellet because the amount of RNA is too small. Here is a link to a thread where I gave my protocol for harvesting RNA from as little as 10 mouse oocytes using trizol
If you are harvesting the oocytes yourself, you can place them directly into trizol and put it in -80 for later use, so RNA later may not be necessary.
http://www.protocol-online.org/forums/inde...&hl=oocytes
I have a problem.. I tried to extract mRNA using Chomczynski method from oocytos in RNAlater.. I added 1 ml of trizol and after shaking and rotating I still could see two phases - It looked like trizol was one phase and RNAlater was a second phase..I continued extraction but I couldn't see any pellet.. I did PCR reaction using One-Step Kit from Qiagen but I didn't see any product..;( (positive control works well..)
I don't have experience with RNAlater. Mostly I extract mRNA directly from human cells seeded on the plate and It works well..
I supposed that something was wrong and I'm still keeping all the supernatants in -80C so I can repeat extraction but I don't know where I did mistake - I don't want to make the same mistake..
please, give me advice what to do.. ( trully I beg for a fast help..)
Aga
Hi... It's me again
Thank YOu for all good advices! It's really useful..
Ok will write what I did step by step..
I got few samples with vary number of oocytos (3 to 11) in RNAlater (100ul to 200ul).. because of small amount of them I was afraid of losing some and I didn't remove Rnalater.. I took my samples to 1.5 ul tube and added 1 ml of trizol. I shook it with hand and I still could see two phases.. So I decided to rotate it in +4 for 10 min. After that I still could see two phases so I shook it longer and I add 100 ul of chlorophorm and mix by shaking with hands. Then I spun it on +4C for 15 min 8000 rpm and removed upper phase to a new tube containing 500 ul of isopropanol. I mixed it by inverting and let it stand on ice for 15 min.. Then I spun it for 15 min 13200 in +4 and removed supernatant. Then I added 1ml of 75% cold ethanol and centrifuge it for 15 min 13200 rpm in +4. After that step I should see the pellet but I didn't. I removed ethanol as much as I could and I add Nuclease Free Water with Rnasin inhibitor and I did RTPCR using OneStep Kit form Qiagen and I didn't get any product. Then I decided to take more potencial mRNA and I did cDNA and PCR for actin and again nothing ;(
I decided to spin the supernatant of ethanol (from the first try - it was in -80C) - I spun it for 30 min at 13200 rpm in +4. I was surprised because on the bottom of the tube I could see some round, bigger and smaller circles ( it didn't look like a mRNA pellet which I used to see after extractiom mRNA directly from the plate - white, attached to the tube).. I removed ethanol and added 30ul of Nuclease free water with Rnasin inhibitor, pippete it well and I tool 2ul for making cDNA, and then actin.. and again nothing ;( now I'm really confused... what was it and where the reason of not getting mRNA is??
I;ll try to take more potential mRNA and did it again ..
Thank You for all answers and usuefull tips - I'm realy waiting for them..
Aga
A few suggestions:
1) I don't know what to tell you about the oocytes in RNAlater. As you have realized, you will probably lose the oocytes when trying to remove it. I would get fresh oocytes and put them directly into Trizol, if possible.
2) I have never tried to get RNA from as little as 3 oocytes. I would use at least 8-10.
3) The isopropanol precipitation should be done overnight at -20 with the additon of glycogen to increase the yield. There's no way you can see the pellet without using glycogen or some other carrier.
4) Only 2 out of 30 ul for RTPCR is way too little. I resuspend the pellet in a small volume and use the entire amount for RTPCR.
Here's the protocol that I use:
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.