small RNA cleanup for Illumina library prep

[eab]

Has anyone tried using Agencourt RNAclean XP beads to purify adapter-ligated small RNAs before the RT step of library prep? Tech support says that 100-base RNAs are not recovered efficiently by their supported protocol, though they are unofficially aware of work-arounds in which smaller molecules are recovered by adding more reagent.

I'm performing 60 library preps in a plate format and would like to avoid running each one over a separate column....

[PigletIA]

RNAclean XP to recover RNA fragment >60nt?

Hi, eab,
I'm facing the same problem now that I want to recover fragmented RNA (using Ambion RNA fragmentation reagent) with size range 60-200nt. Have you figured out a way to do this with the RNAclean XP beads? In my case, I don't care about background binding of smaller oligos since I just want to recover everything.
Thanks!

[eab]

Hi there, I did try RNAclean XP beads at increasing ratios of bead mixture to ligation reaction, up to 3.6:1. Didn't help at all. A company called Zymo makes a 96-well RNA purification column, but the columns didn't perform very well in my hands (difficult to remove residual wash buffer, elution volumes widely divergent between wells). So unfortunately I never came up with a good answer there. Might be worth trying the Zymo if you have a good plate spinner, or at least talking to their tech support about the issue.
Good luck!
Eli

[PigletIA]

Thank you so much for sharing this info, Eli! Have you even tried adding EtOH of isopropanol during the binding to see if it helps? I'll definitely check out the Zymo kit for now
Thanks!
Lan

[eab]

Hi Lan, I never tried that but I see your point - certainly could be worth a try. Would you let me know if it works out?
Eli

[PigletIA]

Absolutely! I just need to wait for the shipment of my beads and maybe another couple of weeks...Or I'm thinking maybe I can play with my AMPure XP beads first since those also have the problem of losing DNA 100nt (or bp? not sure) and below...
Lan

[eab]

Word I had from Agencourt support is that RNAClean beads are just RNAse-free AMPure XP beads.

[PigletIA]

:P I see! Luckily it's the same price per ml, so at least they didn't over-charge it

[pmiguel]

Hi there, I did try RNAclean XP beads at increasing ratios of bead mixture to ligation reaction, up to 3.6:1. Didn't help at all.

Meaning that you did not recover fragments in your range of interest?

You should look at this thread.

The concentration of the precipitator, PEG, in AmPure is probably 7.5%. This may not be a high enough final concentration to pull down very short fragments.

Actually, another thread suggests that even 15% PEG will not work. Going to lower temps, higher salt concentrations and/or longer ppt times may be required.

--
Phillip

[jinpingzhang]

Has anyone tried using Agencourt RNAclean XP beads to purify adapter-ligated small RNAs before the RT step of library prep? Tech support says that 100-base RNAs are not recovered efficiently by their supported protocol, though they are unofficially aware of work-arounds in which smaller molecules are recovered by adding more reagent.

I'm performing 60 library preps in a plate format and would like to avoid running each one over a separate column....

Hi, Everyone,
I think for cleanup RNA small fragment just try XP bead-RNase free,

1. if you have 100ul RNA solution, add:
(1) 3M NaOAc 25ul to final contration 0.3M
(2) Isopropanol 125ul
(3) now total volume is 250ul
2. trans 50ul (=1/5 RNA solution) XP bead into 1.5ml low bind tube
3. set XP bead on magnetic rack for 3 minutes, then discard supernatant.
4. trans 250ul RNA solution to beads tube, mix by pipetting
5. incubate on RT/5min
6. spindown, and put on magnetic rack for 2-3min
7. discard supernatant, keep tube on rack add 70% ethanol ~500ul wash bead by turn down tube several times.
8. discard supernatant, and spindown bead
9. put tube on magnetic rack again, discard residue ethanol.
10. 37degree heat block/~3min, or RT dry bead to no shine
11. add h2o elute your bead, ~20ul.

[pmiguel]

Okay, if I am reading this correctly, the person who designed this method is either hopelessly insane, or some kind of genius. (I guess the latter, if the protocol works.)

Hi, Everyone,
I think for cleanup RNA small fragment just try XP bead-RNase free,

1. if you have 100ul RNA solution, add:
(1) 3M NaOAc 25ul to final contration 0.3M
(2) Isopropanol 125ul
(3) now total volume is 250ul

I read this as a 1:1 Isopropanol precipitation. Traditionally the next step is to centrifuge the tubes and hope the solid phase polynucleotides will stick to the wall of your centrifuge tubes. If you are lucky, and they do, then maybe add a few washed to try to dislodge the metric ton of salt you brought down as well. That is, a cannonical alchohol precipitation.

But instead, jinpingzhang takes -- well I don't even want to call this a "left" turn, because that is a turn anyone would consider. Seems here a turn in a previously unknown direction is made:

2. trans 50ul (=1/5 RNA solution) XP bead into 1.5ml low bind tube
3. set XP bead on magnetic rack for 3 minutes, then discard supernatant.
4. trans 250ul RNA solution to beads tube, mix by pipetting

Okay, if I read this correctly, you are removing the normal Ampure precipitation solution (PEG) from the beads, and instead using an Isopropanol/salt precipitation method but with the same capture media -- the magnetic beads!?!

This works? I mean, I guess there is no reason it wouldn't.

Of course as a fractionation protocol you are swapping the generally benign properties of a PEG precipitation, for the less forgiving properties of an isopropanol precipitation. But the use of beads/magnet instead of centrifugation might be a mitigating factor.

5. incubate on RT/5min
6. spindown, and put on magnetic rack for 2-3min
7. discard supernatant, keep tube on rack add 70% ethanol ~500ul wash bead by turn down tube several times.
8. discard supernatant, and spindown bead
9. put tube on magnetic rack again, discard residue ethanol.
10. 37degree heat block/~3min, or RT dry bead to no shine
11. add h2o elute your bead, ~20ul.

The rest is basically standard Ampure.

Anyone else seen this methodology (alcohol ptt with magnetic bead capture) previously?

--
Phillip

[jinpingzhang]

Okay, if I am reading this correctly, the person who designed this method is either hopelessly insane, or some kind of genius. (I guess the latter, if the protocol works.)


I read this as a 1:1 Isopropanol precipitation. Traditionally the next step is to centrifuge the tubes and hope the solid phase polynucleotides will stick to the wall of your centrifuge tubes. If you are lucky, and they do, then maybe add a few washed to try to dislodge the metric ton of salt you brought down as well. That is, a cannonical alchohol precipitation.

But instead, jinpingzhang takes -- well I don't even want to call this a "left" turn, because that is a turn anyone would consider. Seems here a turn in a previously unknown direction is made:


Okay, if I read this correctly, you are removing the normal Ampure precipitation solution (PEG) from the beads, and instead using an Isopropanol/salt precipitation method but with the same capture media -- the magnetic beads!?!

This works? I mean, I guess there is no reason it wouldn't.

Of course as a fractionation protocol you are swapping the generally benign properties of a PEG precipitation, for the less forgiving properties of an isopropanol precipitation. But the use of beads/magnet instead of centrifugation might be a mitigating factor.



The rest is basically standard Ampure.

Anyone else seen this methodology (alcohol ptt with magnetic bead capture) previously?

--
Phillip

Hi, Phillip,
I design this method base on properties of some chaotropics, like Ethanol, isopropanol,.. selection of those chaotropics let us change the range of purity flexible. for save beads, for save time and money, gain your wanted results.

Jinping Zhang at MDACC

[pmiguel]

Hi, Phillip,
I design this method base on properties of some chaotropics, like Ethanol, isopropanol,.. selection of those chaotropics let us change the range of purity flexible. for save beads, for save time and money, gain your wanted results.

Jinping Zhang at MDACC

I have to say that I like the way you think.

As to the method: the reason to use an acrylamide gel, is that it seems hopeless to distinguish between 121 bp (adapter dimers) from 140 bp (about the minimum size of adapters (121 bp) + small RNA insert (~19 nt). Does you method successfully resolve these two types of products?

--
Phillip

[jinpingzhang]

Ok,
we have three method for purification or concentrate the NA,
1) precipitation
2) bead
3) column

precipitation will get the highest yield ratio, but time consumable, at lest 1hr.
1) 50ul RNA soln, add 18ul 5M NH4OAC, add 111ul 100%EtOH, 20ug Glygogen(~4ulx5ug/ul) total ~184ul.
2) gentally mixwell and highspeed/40min/at 4 degree.
3) ~300ul 70% EtOH wash pellet.
4) air dry pellet/~10min, elute in your required ul H2O
column will get highest purity, but dont use more volume and high salt. regularly, increase 100ul vol will decrease yield ratio ~6%, even with same concentration salt and chaotropic agent. for example, if transfer 200ul to column, finally get ~80% inputting RNA. use precipitation method just no Glycogen.
bead method will get the higher yield ratio less than precipitation, but maybe have some residual Wash Buffer( EtOH), Will affect down strem reaction.
My suggestion is use precipitation or column with no more than 200ul volume per column or tube.

[pmiguel]

Hi jinpingzhang,

Sure, for purification/concentration. But what about size selection? Would any of those work to select 140-165 bp fragments away from 121 bp fragments?

--
Phillip