8 replies to this topic

[lab_member]

I tried using the RNeasy Plus micro Kit (claims it can isolate RNA from a single cell) at first to extract RNA from ~20,000 cells (human ES cells) but I only got a very low yield of 22 ng/ul (14 ul elution volume)

The profile of the absorbance (after reading in nanodrop) has a peak at ~230 nm but a very low peak at 260 nm. The 260/230 value is only 0.31 while the 280/260 value is 1.71.

Today, I tried to use the RNEasy Mini Kit (claims it can isolate RNA from min of 100 cells) to extract RNA from ~40,000 cells (human ES again) and only got 10 ng/ul (30 ul elution volume). Similarily there was a peak at 230 nm but no peak at 260 nm.

Does anyone know what went wrong and which are the steps that is especially important or some tips on getting higher yield.

Thanks  

[Curtis]

in our lab we prefer to use Trizol method because Qiagen's RNeasy kit gives low yield. but on the other hand our Qiagen's RNA has better quality and we can use it for Real-Time RT-PCR.

personally I think filter based RNA extraction methods don't give high yield because still a lot of RNA remains in the filter. I would still prefer pellet based methods with iso propanol.

but, just one question? did you add the 30 ul of elution buffer in the last step to the center of the filter? did you leave it for a while before centrifuge?

Edited by Curtis, 25 March 2009 - 03:55 AM.

[jah]

In my experience with RNeasy Mini Kits, I've come up with a couple of extra steps (not in their protocols) that increase yield and purity.
1) Make sure to use B-ME in the RLT lysis buffer(optional in the current protocol), and make the 70% EtOH fresh. (increased yield)
2) After applying the RLT-lysate to the column and centrifuging/vac'g run a second bolus of fresh RLT through the column (helps decrease 280nm contamination)
3) Perform an extra wash with RW1 (helps decrease 280nm contamination)
4) Mix the RPE stock and EtOH fresh(1:4);  Perform an extra wash with RPE (helps decrease 280 and 230 peaks)
5) Allow the columns to air dry & evaproate EtOH with the caps open for ~5min before elution (increases yield and decreases 230 peak)
6) Pre-warm the water (~45`C) before elution.  As Curtis said, add the water to the center of the membrane, and let it sit for 2-3 minutes (increases yield)

6a)If you have a SpeedVac, you can increase your yield by increasing your elution volume to 100ul, and also do one more step
7) After the first elution, transfer the RNA to hold on Ice.  Repeat step 6.  Pool the eluents (~190ul total) and concentrate in Speedvac ~30`C/max vacuum for ~10-20 minutes.  Just be careful not to over-dry; I check the tube at 10, 15 and 20 minutes, and stop when I have ~10-15ul concentrate.   This will also evaporate any residual EtOH part of the 230 peak

I've only recently added #'s 6a and 7 to my protocol, but I don't seem to have problems with degradation, and consistently get 260/280's >1.85.  Most of these modifications came from suggestions made on Bioforum regarding various experiences with Qiagen DNA columns.  Most seem to translate pretty well for the RNA methods as well. Good Luck!

Edited by jah, 25 March 2009 - 06:03 AM.

[lab_member]

Curtis, on Mar 25 2009, 07:54 PM, said:

but, just one question? did you add the 30 ul of elution buffer in the last step to the center of the filter? did you leave it for a while before centrifuge?

Hi Curtis,

I did add the 30 ul elution buffer (which is actually RNase free water provided) to the center of the filter, then I leave it for like 5 min before I elute, but the yield was low as in my first post.

Actually, I had always thought that the Trizol method is unable to pellet enough RNA for a small number of cells which is why I chose the column method... someone else in my lab tried Trizol with more cells than I had but did not manage to extract enough RNA (low yield, bad looking curve on nanodrop)

Thanks for your suggestion :-)

[lab_member]

jah, on Mar 25 2009, 09:56 PM, said:

In my experience with RNeasy Mini Kits, I've come up with a couple of extra steps (not in their protocols) that increase yield and purity.
1) Make sure to use B-ME in the RLT lysis buffer(optional in the current protocol), and make the 70% EtOH fresh. (increased yield)
2) After applying the RLT-lysate to the column and centrifuging/vac'g run a second bolus of fresh RLT through the column (helps decrease 280nm contamination)
3) Perform an extra wash with RW1 (helps decrease 280nm contamination)
4) Mix the RPE stock and EtOH fresh(1:4);  Perform an extra wash with RPE (helps decrease 280 and 230 peaks)
5) Allow the columns to air dry & evaproate EtOH with the caps open for ~5min before elution (increases yield and decreases 230 peak)
6) Pre-warm the water (~45`C) before elution.  As Curtis said, add the water to the center of the membrane, and let it sit for 2-3 minutes (increases yield)

6a)If you have a SpeedVac, you can increase your yield by increasing your elution volume to 100ul, and also do one more step
7) After the first elution, transfer the RNA to hold on Ice.  Repeat step 6.  Pool the eluents (~190ul total) and concentrate in Speedvac ~30`C/max vacuum for ~10-20 minutes.  Just be careful not to over-dry; I check the tube at 10, 15 and 20 minutes, and stop when I have ~10-15ul concentrate.   This will also evaporate any residual EtOH part of the 230 peak

I've only recently added #'s 6a and 7 to my protocol, but I don't seem to have problems with degradation, and consistently get 260/280's >1.85.  Most of these modifications came from suggestions made on Bioforum regarding various experiences with Qiagen DNA columns.  Most seem to translate pretty well for the RNA methods as well. Good Luck!

Hi Jah,

thanks for your suggestion! I think I'll try it today! I hope I get some RNA.

[Curtis]

lab_member, on Mar 25 2009, 08:18 PM, said:

Hi Curtis,

I did add the 30 ul elution buffer (which is actually RNase free water provided) to the center of the filter, then I leave it for like 5 min before I elute, but the yield was low as in my first post.

Actually, I had always thought that the Trizol method is unable to pellet enough RNA for a small number of cells which is why I chose the column method... someone else in my lab tried Trizol with more cells than I had but did not manage to extract enough RNA (low yield, bad looking curve on nanodrop)

Thanks for your suggestion :-)

sometimes I use 3 ul of virus sample to get RNA and it works fine. So I think it shouldn't be a problem if you are careful when you are collecting the top phase of Trizol-Chloroform. but you usually don't see the RNA pellet when you centrifuge with Iso-p for that amount of low titre virus.

[aimikins]

just switch to Stratagene's kit.  it's cheaper, cleaner, and has a better yield.

[lab_member]

Hi Jah,

thanks for your methods. I managed to get sufficient RNA though I got some 230 nm contamination (I tried to precipitate and wash with 70% ethanol) but I just continue on. I really think it is a very good method and I am going to suggest this method to some of my lab colleagues who are also going to do RNA extraction too! To be frank, I actually used the PureLink kit instead of RNeasy to follow your methods (the reagents are actually the same I guess, because the protocol is almost the same).  


To Curtis: My senior also told me that it is very skill-dependent to extract the to phase...so I guess my skills still need a lot improvement...but she says it is the best method (if your skill is good)


To aimikins: True that Qiagen RNeasy kit is not the best. I had always thought Qiagen is good with columns. My lab members tested and extracted some RNA from fibroblast using both PureLink (Invitrogen) and RNeasy (Qiagen) and we realized PureLink gave cleaner RNA --> much less 230 nm contamination. Is stratagene cheaper than PureLink too? too bad I can't actually decide what kit to buy, I just use what is available in the lab...lol...

Edited by lab_member, 27 March 2009 - 05:30 PM.

[lab_light]

jah, on Mar 25 2009, 06:56 AM, said:

In my experience with RNeasy Mini Kits, I've come up with a couple of extra steps (not in their protocols) that increase yield and purity.
1) Make sure to use B-ME in the RLT lysis buffer(optional in the current protocol), and make the 70% EtOH fresh. (increased yield)
2) After applying the RLT-lysate to the column and centrifuging/vac'g run a second bolus of fresh RLT through the column (helps decrease 280nm contamination)
3) Perform an extra wash with RW1 (helps decrease 280nm contamination)
4) Mix the RPE stock and EtOH fresh(1:4);  Perform an extra wash with RPE (helps decrease 280 and 230 peaks)
5) Allow the columns to air dry & evaproate EtOH with the caps open for ~5min before elution (increases yield and decreases 230 peak)
6) Pre-warm the water (~45`C) before elution.  As Curtis said, add the water to the center of the membrane, and let it sit for 2-3 minutes (increases yield)

6a)If you have a SpeedVac, you can increase your yield by increasing your elution volume to 100ul, and also do one more step
7) After the first elution, transfer the RNA to hold on Ice.  Repeat step 6.  Pool the eluents (~190ul total) and concentrate in Speedvac ~30`C/max vacuum for ~10-20 minutes.  Just be careful not to over-dry; I check the tube at 10, 15 and 20 minutes, and stop when I have ~10-15ul concentrate.   This will also evaporate any residual EtOH part of the 230 peak

I've only recently added #'s 6a and 7 to my protocol, but I don't seem to have problems with degradation, and consistently get 260/280's >1.85.  Most of these modifications came from suggestions made on Bioforum regarding various experiences with Qiagen DNA columns.  Most seem to translate pretty well for the RNA methods as well. Good Luck!

Hi Jah,
Thank you so much for sharing all this great info.  I had a couple of  questions:
step #2
"2) After applying the RLT-lysate to the column and centrifuging/vac'g run a second bolus of fresh RLT through the column (helps decrease 280nm contamination)"
do you just add another 350ul of RLT with BME without anything through the column? and if so why do you think this would decrease the 280 contamination.
and what kind of starting marterial are you working with,
as to put my two cents, my lab  collegue and I when we compared our starting materials we realized the importance of the starting material as  bacteria as a starting material are much more tough than mammalian tissues while cell lines are tougher than primary cells for isolation of RNA, and so on. did you have the same experience.
Thanks,
lab_light