Hi Scicong

1. After you (preferably) vortex your pellet (plus glycogen) or if pellet small and fairly inconspicuous so loss of pellet by dislodging is a worry and thus you repeatedly and gently pipette up and down your room temp ethanol, simply removing your ethanol and allowing the air pellet to air dry is in my experience sufficient to raise the 260/230 ratio without incurring loss of too much RNA. This incidentally is another reason for adding glycogen to your RNA in addition to 1/10 3M sodium acetate plus 3 x volumes of ethanol when you precipitate. In the presence of glycogen you will recover more than 90% of your RNA and this effect is particularly beneficial when precipitating 1ug or less (a nd I think in fact beneficial up to 10ug of total RNA).

Thus, to go back to your question simply washing x 2 and air drying your pellet is enough to render your 260/230 ratio > 1.0 which is where you want it to be

2. That said if you did precipitate and then put down a column for a second time you would end up with very clean RNA indeed - in fact the highest 260/230 and 260/280 ratios of all - but I WOULD NOT recommend this procedure unless you precipitate with glycogen and not if you expect to obtain just a few ug of total RNA in yield: In this situation you might find you obtain very clean RNA but < 1ug in total which is what ideally you need for a consistent and efficient RT reaction

3. With regard to your second question which concerns the alternative method, namely column purification; taking eluate mixing with 70% ethanol and then performing a second column purification with no precipitation between the 2 then having loaded onto the column you then carry out a standard RNEasy purification but as mentioned with a 1 minute wait after adding the 70% ethanol based wash buffer; repeating this addition, wait 1 minute them spin again and waiting 5 niutes before eluting RNA and then putting that first pass eluate back onto the column; waiting a further 5 minutes and then spinning again

Going back to your exact question, this protocol is obviously designed so that no ethanol is finally eluated with your aqueous based (water or TE) RNA: Specifically, before adding water or TE there is a spin step where you subject the column to a 1 minute spin at maximum speed before adding elutant, waiting 5 minutes and eluting your RNA. This spin is designed to remove any remaining ethanol based wash buffer from the column - in other words dry the column - before adding your elutant

When performing  this step make sure you perform a longer (3 minute spin) @ 10K (in a microfuge) with second ethanol based wash before spinning your column for 1 min at maximum speed to remove any residual ethanol. Indeed, all ethanol should be removed during the prior 3 minute spin (after adding 2nd lot of wash buffer) so that when you spin at maximum speed for 1 minute you shouldn't see anything else elute. Thus, the real purpose of the 1 minute spin is simply to confirm that the column is already dry (and thus no ethanol can contaminate your RNA). If you do see a tiny amount of liquid eluated, then repeat this 1 minute spin. Very occasionally I have had to do this and with a second 1 minute spin your column will absolutely be dry. But to reiterate, with a 3 minute spin @ 10k it is rare for more liquid to come out during the 1 minute spin. If it des by the way that usually implies a partly blocked column and thus purification complications and in this situation the best solution is to start again

Hope this helps and do not hesitate to contact me with further questions

Hello Sicong

Yes I have experienced what you are talking about: At concentrations of RNA >/= 50ng/ul, you obtain a nice peak @ 260nM and the 260/230 ratio is sensible and reflects guanidine/trizol contamination

At the sorts of values you are talking about where the absorbance peak is not regular I have noticed odd 260/230 ratios; In these instances by ethanol precipitating the sample and deliberately resuspending in a much lower volume to increase concentration the absorbance peak becomes a well defined peak and the 260/230 ratio is > 1.0, which is what you want to not have to worry about the effects of Trizol and GITC on downstream enzymes like RT

To increase the 260/230 ratio to > 1.0 you van either:

1. Ethanol precipitate as you suggest

2. Alternatively, take the eluate from your column add an equal volume of 70% ethanol and put down the column again

Both methods have worked effectively in my hands: See below

Ordinarily in my hands ethanol precipitation has fixed the issue. However, I would strongly suggest performing 2 x ROOM TEMP 70% ethanol washes to desalt the pellet and remove any residual GITC. Furthermore, if you can add 1ul of 1mg/ml to 20mg/ml glycogen to the pellet (as an inert carrier) that will render the pellet larger and more opaque and allow you to vortex the pellet, having removed ethanol spt after precipitation and added the 70% ethanol, which will increase the efficacy of your desalt wash (in the absence of glycogen you can still wash/desalt with room temp 70% ethanol by vortexing but you need to pay attention to where you smaller less opaque pellet resides in the tube as as not to lose)

I would also add that taking eluate from the column; adding an equal volume of 70% ethanol and then putting down a fresh column for a second time can also increase the 260/230 ratio

Finally for first and second (if necessary) column purifications to increase the 260/230 ratio at the outset and increase the RNA yield such that you don't experience artifactual readings link to skewed absorbance curves owing to poor concentrations on the nanodrop you can (respectively):

1. Add your second ethanol based buffer to the column and then WAIT 1 MINUTE before spinning through; and then repeat this second ethanol based buffer wsh: This wash is designed to desalt the column (generally synonymous with removal of guanidium salts) and leaving for 1 minute before spinning through simply allows the ethanol based buffer to insinuate more effectively into the dead spaces between the silica fines in the column and dissolve salt, thus leading to more effective salt removal when you then spin that buffer out

2. Add 30ul of water or TE to dry column when coming to elute WAIT 5 minutes; spinning for 1 min at full speed then putting the eluate back onto the column; waiting 5 minutes as before and then finally spinning through

mirVana spin column based kit works great for plasma/serum, or for high throughput- MagMax mirVana. higher RNA yields, high purity 260/230, 260/280

Hi Laurence, 

Thank you so much for your detailed response! A couple of questions:

After you perform your EtOH precipitation and 70% EtOH wash, do you do a cleanup with a column or just take off supernatant & let your pellet air dry?

In your advice to add equal volume of 70% EtOH to the eluate, then spin again in a new column - how will the EtOH be filtered out of my eluate? 

Hi Scicong

1. After you (preferably) vortex your pellet (plus glycogen) or if pellet small and fairly inconspicuous so loss of pellet by dislodging is a worry and thus you repeatedly and gently pipette up and down your room temp ethanol, simply removing your ethanol and allowing the air pellet to air dry is in my experience sufficient to raise the 260/230 ratio without incurring loss of too much RNA. This incidentally is another reason for adding glycogen to your RNA in addition to 1/10 3M sodium acetate plus 3 x volumes of ethanol when you precipitate. In the presence of glycogen you will recover more than 90% of your RNA and this effect is particularly beneficial when precipitating 1ug or less (a nd I think in fact beneficial up to 10ug of total RNA).

Thus, to go back to your question simply washing x 2 and air drying your pellet is enough to render your 260/230 ratio > 1.0 which is where you want it to be

2. That said if you did precipitate and then put down a column for a second time you would end up with very clean RNA indeed - in fact the highest 260/230 and 260/280 ratios of all - but I WOULD NOT recommend this procedure unless you precipitate with glycogen and not if you expect to obtain just a few ug of total RNA in yield: In this situation you might find you obtain very clean RNA but < 1ug in total which is what ideally you need for a consistent and efficient RT reaction

3. With regard to your second question which concerns the alternative method, namely column purification; taking eluate mixing with 70% ethanol and then performing a second column purification with no precipitation between the 2 then having loaded onto the column you then carry out a standard RNEasy purification but as mentioned with a 1 minute wait after adding the 70% ethanol based wash buffer; repeating this addition, wait 1 minute them spin again and waiting 5 niutes before eluting RNA and then putting that first pass eluate back onto the column; waiting a further 5 minutes and then spinning again

Going back to your exact question, this protocol is obviously designed so that no ethanol is finally eluated with your aqueous based (water or TE) RNA: Specifically, before adding water or TE there is a spin step where you subject the column to a 1 minute spin at maximum speed before adding elutant, waiting 5 minutes and eluting your RNA. This spin is designed to remove any remaining ethanol based wash buffer from the column - in other words dry the column - before adding your elutant

When performing  this step make sure you perform a longer (3 minute spin) @ 10K (in a microfuge) with second ethanol based wash before spinning your column for 1 min at maximum speed to remove any residual ethanol. Indeed, all ethanol should be removed during the prior 3 minute spin (after adding 2nd lot of wash buffer) so that when you spin at maximum speed for 1 minute you shouldn't see anything else elute. Thus, the real purpose of the 1 minute spin is simply to confirm that the column is already dry (and thus no ethanol can contaminate your RNA). If you do see a tiny amount of liquid eluated, then repeat this 1 minute spin. Very occasionally I have had to do this and with a second 1 minute spin your column will absolutely be dry. But to reiterate, with a 3 minute spin @ 10k it is rare for more liquid to come out during the 1 minute spin. If it des by the way that usually implies a partly blocked column and thus purification complications and in this situation the best solution is to start again

Hope this helps and do not hesitate to contact me with further questions