[PStepper]

Hi everyone,

this is the main discussion space for BOMB protocol #6.4 TNA extraction from plants.
Here you can post all your questions and feedback!

[PStepper]

reserved space

[Mike Axtell]

Has anyone examined the recovery of microRNAs (~21 nt RNAs) using this TNA protocol, or with any of the BOMB protocols?

[Phil Oberacker]

Hey Mike, sorry for the late answer!

We have indeed not done this for RNA as small as microRNAs. However, the binding of DNA as well as for RNA to the silica-beads depends on the salt concentration during the binding step. For DNA we recently developed a protocol for nucleotide removal (#4.4), where we capture DNA oligos with a length of about 25 nts with a yield of ~30%. The trick was to increase the salt concentration, by using 5- to 10-fold more binding buffer (10 or 20 volumes compared to 2 volumes) and therefore, higher salt concentrations. I would assume this could work for RNA as well, if one increases the amount of binding buffer in #6.3. However, neither of us has tested this yet. Maybe give it a shot and let me know how it went for you.

Cheers, Phil

[Jstees]

Does anyone else have an issue where a white precipitate forms with the addition of the GITC on step 3?

I am wondering if the precipitate is the SDS or the GITC.

[TimH]

Hi Jstees – apologies for the slow reply! Yes, when adding GITC following TNES incubation (i.e. the DNA only tissue protocol) there is formation of precipitate, presumably SDS. Nevertheless, in our hands this clarifies at the isopropanol stage and does not appear to adversely affect purification. But please let us know if you find otherwise. Best wishes, Tim

[Merrin Spackman]

Hi

I am doing large scale extractions (1g) from wheat where I need large volumes of lysis buffer (6 ml min).  In your protocol you separate the TNES and GITC buffers.  Is it critical to have the high salt in the buffer and can I add that to the GITC buffer so I only use one buffer.

 

thanks

Merrin

[Phil Oberacker]

Hi Merrin,

In the step before the addition of GITC you do a proteinase K and RNase A digest (depending on what you want to extract, TNA or RNA). GITC in general denatures proteins. While proteinase K activity can actually be enhanced at lower molarities, I don’t know how it will effect the enzyme a the concentrations used in the protocol (4-6 M). The bigger issue, however, will be the RNase A as it will be denatured for sure. But then again, we haven’t tried out adding the GITC to the digest. Maybe give it a try and let us know how it worked out for you.

Cheers
Phil

[Mike Axtell]

Hi,

I’m seeing strong absorbance at ~ 220-230 nm with TNA from this protocol (6.4). Suspect it is carryover of GITC. Also noticing that my silica beads tend to clump a little during binding, and only seem to “de-clump” upon elution. Wondering if the two observations (Guanidinium carryover and bead clumping) are connected, but even if unconnected, what I’d really like to solve is the carry-over of GITC into the final TNA eluate.

 

Thanks!

 

[Phil Oberacker]

Hi Mike,

indeed the absorbance at 220-230 nm is most likely GITC and yes, the two observations you made are probably connected. We usually see this clumping behaviour of beads when they are overloaded with nucleic acid. I posted an update addressing this on Researchgate (Project log Feb. 26: https://www.researchgate.net/project/Bio-On-Magnetic-Beads-BOMB). The flakes or clumps can obviously not be washed as properly as free beads leading to carryover of GITC and other things. I would recommend to either use less of the sample or more beads (maybe both). Let us know how it works for you.

Cheers,
Phil

[Mike Axtell]

Thanks Phil, I appreciate your advice. I tried a series of extractions holding tissue amount constant (~ 18mg Arabidopsis leaves) and increasing silica bead usage over a ten-fold range. More beads indeed reduced clumping. However, the strong GITC contamination in the eluted TNA was not affected at all by the bead amounts.

I also did the same experiment (varying bead amounts) in mock extractions, with no plant tissue at all, with the same result: Very strong levels of GITC (inferred from absorbance 220-230nm) in all conditions, in the absence of any biological input.

I’m following the 6.4 protocol quite closely, and I remade all solutions in this most recent trial. The only small difference is that I use “LTE-7.5” to elute the TNA, instead of water (LTE-7.5 is 10mM Tris-HCl pH 7.5, 0.1mM EDTA). The silica-coated beads I am using were synthesized according to the BOMB protocol, and everything seemed as expected : good yield, no evidence of oxidation upon storage.

Any other suggestions?

Thanks!,

Mike Axtell

[Phil Oberacker]

Hi Mike,

Great so at least the clumping issue is dissolved then (pun intended). Did you see any increase in yield, when adding more beads?

I don’t think the elution buffer is causing the GITC takeover. However, you could compare it by running a sample and elute with pure ddH2O, though, and see if that changes things. But as I said, I don’t think the buffer is the problem. How many times do you wash with ethanol? You could try to add two more washing steps. I usually do this with the RNA protocols and never experienced any takeover of GITC.

You mentioned that you’re following the animal tissue protocol #6.3. We also have a protocol specifically designed for plant tissue #6.4 which might suit your application better. However, the protocols are quite similar and only a few steps are different or swapped. As these two protocols were designed by our NZ team, I’m personally not too familiar with them. However, as only the initial steps are different and you get a good yield, I think it doesn’t make a big difference here. You might want to have a look, though.

Let me know if additional washing steps help you get rid of the GITC. If you still have troubles, I’ll forward your issue to our NZ team who are more familiar with TNA isolation from plant tissues.

Cheers,
Phil

[Phil Oberacker]

PS: How do you wash your beads? Do you fully dissolve them? If you pipette them up and down or even vortex them, you should be able to have them nicely dissolved, which definitely increases the efficiency of the wash step.

[Mike Axtell]

Thanks Phil, I really appreciate your time and advice!

I’m using the plant GITC TNA protocol 6.4. Clicking the “forum” link for protocol 6.4 leads to this forum (but I did notice the forum itself says it’s for 6.3). Sorry for confusion there.

Getting more aggressive with bead washing did help. Before I was just tumbling and flicking by hand. In my latest trial I pipetted up and down aggressively, and gave a brief vortex. I also upped the volume of 80% ethanol used to wash, and did four washes instead of two.

That did reduce the absorption at ~220-230nm, by quite a bit, but I still have not so good 260/230 ratios .. around 1.0 to 1.2. Nucleic acid yield is about what I expect given the tissue inputs I am using. Just wish I could get it even cleaner. I’ll keep running down some other variables and post again if I manage to fully solve it. And yes if your NZ colleagues had any ideas I would welcome them.

Some googling led me to fact that Iron(III) and maybe other Iron compounds also absorb down there in the 220-230nm range. I wonder if residual nanoparticles or free Iron is it?

Thanks again!

Mike

 

 

[Phil Oberacker]

Hi Mike,

No worries, glad if I can help!

Good to know! I will forward that to our webpage manager! That should not be the case.

Great! Glad the thorough washing did improve the quality of your samples. I agree, these ratios don’t sound good. However, as I personally never used the plant protocol myself, I’m not sure what to expect. Plants generally contain tons of carbohydrates and phenolics which might stick to the beads more aggressively, and both could lead to 230 peaks as well as far as I know. I’m not sure about ironIII though. Although it might cause such a peak, I doubt it would be carried over as it should be oxidized quite quickly. I found this nice PDF online showing different contamination graphs for NanoDrop. Does any of these look like your samples?

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&ved=2ahUKEwjzm5GSpsLqAhUoxaYKHaVDDnEQFjABegQIARAB&url=https%3A%2F%2Fwww.researchgate.net%2Fprofile%2FZx_Chong%2Fpost%2FLow_260_230_ratio%2Fattachment%2F5c74a938cfe4a781a5834be0%2FAS%253A730387542179840%25401551149368146%2Fdownload%2Fnanodrop.pdf&usg=AOvVaw0B4Vj3HCnv0-jMB2FDokNw

I will anyway forward this to the guys who developed this protocol and ask what their samples looked like in regards to purity. Maybe they can help shining some light on this matter.

Cheers,
Phil

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