Pre- and Post-mRNA fragmentation electropherograms

[grassgirl]

Hi,

I'm new at sequencing and am using the Roche cDNA rapid library prep for 454 Junior sequencing. I fragmented my mRNA as directed using zinc chloride and ran pre- and post-fragmentation samples on the Bioanalyzer pico chip 600. Just wondering if the fragmentation is complete enough. There are spikes at around 150 and 300 nt but still a broad bump peaking at around 2000 nt. I'm attaching the image file.

[grassgirl]

I should have added that the first electropherogram is the unfragmented mRNA and the second electorphergram is the fragmented mRNA

[vlee2]

Seems that fragmentation is not complete. Normally if we see large spikes in small area of the original mRNA, we have them also after fragmentation. But you don't have these spikes in the original mRNA.
If you have problems with getting more mRNA for new procedure, I'd continue with cDNA preparation, but in the end you will have to get rid off large fragments. I usually use about 50% of AmPure beads to bind only large DNA.

[grassgirl]

vlee2, thanks for the suggestion. I went ahead with cDNA prep and in the Rapid Library Prep the next step is purifying with AmpPure beads. Will this step get rid of large fragments or are you speaking of somewhere else in the process? Also, when you say 50% do you mean 50% of total volume of cDNA?

[cystron]

Mysterious Agilent Trace

It appears in these two agilent traces that the sample got bigger during the fragmentation process if the first one is unfragmented and the second is fragmented.
One or both lanes of the agilent mis-behaved.
I believe the other user is recommending a two step SPRI like that which is used in the 454 PE protocol which removes excessively large and excessively small fragments leaving only the mid-range fragments from your orginal library.

I always prefer to run a DNA chip on the dsDNA library rather than rely on trusting the RNA chip

[grassgirl]

cystron,

Thank you for your reply. Yes, I have to wonder about the behavior of the bioanalyzer run. We decided to continue on but to use the nebulizer to fragment the cDNA, then quantitate it and check it for quality on the DNA chip. There were fragments of the desired size, but my quantity ended up being too low to continue, so it's back to the drawing board from the total RNA step.

I will have to seek out the 454 PE protocol for the two-step removal process for future libraries.

[RCJK]

Hi Grassgirl, what time and temperature did you use for your fragmentation? I tried the 70 degrees C for 30 seconds as in the protocol and it didn't do much to my samples. I've now been trying to optimize this step, trying 60, 90, 120, 150, 180 seconds and am getting inconsistent results coupled with weird bioanalyzer traces that make comparing the fragmentation conditions not so straightforward.

If you or anyone else has any suggestions for this step of the protocol, please let me know! I do not have much sample to play around with, so I am experimenting with different samples and am trying to obtain a roughly consistent fragmentation.

Thanks!

[cystron]

More Consistent Fragmentation

the Time and temperature are not as important to the fragmentation as the pH of the buffer used to make the ZnCl solution.

[grassgirl]

RCJK, I followed the protocol in the Roche cDNA Rapid Library Prep Method Manual - just like you I did 70 degrees for 30 seconds. I was thinking of trying an optimization experiment as well, but from your post it is not very encouraging. One thing I thought might be a problem is that I precipitated my RNA prior to fragmentation using NH4OAC and thought that this time I'd try to precipitate it with NaOAC instead, in case that is affecting the reaction.

As for the pH of the buffer used to make the ZnCl, I am also following the protocol and using 1M Tris HCl pH 7.0.

[RCJK]

Thanks for the info. I've made the ZnCl solution (and all other solutions) as per the Roche protocol. I just am finding that 30 seconds is not nearly enough. I think ~125 seconds seems to be working for me. Some of the fragments are smaller than I'd like, but when I tried with less time, there were still too many large fragments present.

[grassgirl]

That's great information RCJK. I'm going to do an optimization tomorrow and was wondering which time points to hit, so I'll shoot for the 125 seconds to see how that goes.

By the way, what organism and tissue are you optimizing for?

Thanks!

[grassgirl]

Just thought I'd add an update on mRNA fragmentation. After doing an optimization experiment, I found that it helped to not only increase incubation time, but to also increase the pH of the fragmentation buffer. For my sample, 120 second with a buffer of pH 8.0 worked the best in an experiment that included the fragmentation buffer at pHs 7.0, 7.5, and 8.0 with 2 time variables for each: 30 seconds and 120 seconds. I'm including the Bioanalyzer traces in case this is helpful to others. The conditions are as follows:

VHU1 and VHU2: Unfragmented reference samples
VHF1: pH 7.0, 30 seconds
VHF2: pH 7.0, 120 seconds
VHF1: pH 7.5, 30 seconds
VHF1: pH 7.5, 120 seconds
VHF1: pH 8.0, 30 seconds
VHF1: pH 8.0, 120 seconds

[bfuente]

problems with RNA fragmentation

hello
We're working followed the Roche protocol to obtain cDNA Librarys but we can't fragment the mRNA. We need obtain fragments between 450-1200 bp. We work with Tris pH7 to do the frag solution and zinc chloride. Incubation 70ºC-30 sec, but the size of mRNA is the same at the end than the begining. Someone can help us? We need work with other pH or change the temperature and time to obtain mRNA fragments?
Thanks

[grassgirl]

bfuente, try increasing your incubation time and/or pH of the buffer. See my previous message on an experiment I did with various pH and times. I found that I need to incubate my samples for 2 minutes. You can try this with the pH at 7.0, or try changing both pH and time.

Good luck.

[RCJK]

I agree w/ grassgirl, I have found ~90-120sec to be good a good incubation time. I have no idea how they think 30sec is enough.