Uneven fragment population Truseq mRNA

[robertami]

Hi there.

Starting off by thanking you all for sharing your experiences in this forum, that's been really helpful!Sorry if this topic has been covered already but I could work out my problem so far reading other posts so there you go.

I'm having a bit of a problem in interpreting my Bioanalyzer trace on a cDNA library made with the Truseq mRNA kit. My RNA RIN was 9 so perfect I guess, then I've used 0.5,1 and 2 ug of RNA input (the figure shows just the result for the 0.5 ug input though), fragmentation time 8 minute which should give a fragment lenght of 300 bp (with adapters). All purification steps done with Ampure beads. In general I've followed the Illumina protocol religiously, no kiddin'. Then I quantify the library with either nanodrop (which gave me around 70 ng/uL for the three samples) and then with Qubit that gave me around 39-42 ng/uL. Never use nanodrop again!
I am quite surprise to see such a small pick compared to most of the other Bioanalyzer traces you all have shared so far. Also what surprise me most is too see such a wide range of fragment, resulting in a bump around 600/700 bp.
Have you had any experience of that at all? I've done a lot of work optimizing real time PCRs and I know how easily the amplification reaction can be inhibited by too much input, so I was wondering whether that's what is actually happening?
Any idea, suggestion, comment would be enormously appreciated!
Thank you all

Roberta

[robertami]

PS: I've run 15 cycles of PCR as indicated in the protocol

[kwaraska]

Have you considered that it might be bubble PCR products from too many cycles?

If I recall 15 is the upper limit of cycles so fewer may actually give you more height in the size range you are shooting for.

[robertami]

Thanks kwaraska for your reply. It actually is bubble product as Illumina tech support confirmed. Wondering why they suggest 15 cycles as default number of cycles to use if that's the upper limit!
Trying to lower this number now, I'll post the results next week thanks a lot

[robertami]

Hello,

Following up from last time I've decided to carry on with 2ug RNA input.

The first issue I faced las time was bubble product (slide 1 and 2 of the attached file)
I think that the bubble product issue has been resolved do you agree?

Now I am experiencing few issue in trying to get 200 bp insert (400 BP with the adapters) See slide 3 and 4 bioanalyzer traces.

I've tried to follow the Illumina truseq stranded protocol so I either left the sample at 4C for a couple of minute during the fragmentation step or incubate at 94C the sample for 1 minute.

I have three questions, hopefully some experienced users may help me out:

1) Slide 3: would you say this is what I should expect from 1 minute 94C fragmentation? If yes, why I have such a wide range of fragments sizes?

2)Slide 4: would you say this is what I should expect from 0 minute 94C (2 minute 4 C) fragmentation? The spiky profile of the trace really worries me.

3) If you have a careful look at the traces, they all show a peak at really high bp, right where the upper marker is.
Is this what I should expect after Ampure XP size selection? I was sure it was a really strong way to size select the fragments. Or it is due to an incomplete fragmentation? Or too much RNA Input perhaps?

Thanks you in advance for your replies!

A desperate PhD student

[pmiguel]

You need to factor out a couple of critical parameters for this to make sense:

(1) Obviously, fragmentation time -- the shorter it is, the longer your maximum cDNA size will be.

(2) Just as importantly, Ampure cuts need to be adjusted to remove any cDNAs that are below your intended cut-off size.

I think you can draw the correct conclusions about your results using these two considerations.

Well, with the stranded kit there are a couple of additional issues:

(3) First strand synthesis time dropped from 50 minutes in the non-stranded kit to 15 minutes in the stranded kit. This may be limiting the maximum cDNA length.

(4) This one is more speculative, but I am concerned enough to mention it here. I think it is possible that SuperScript II does not do well frozen solid with the "First Strand Synthesis Mix". Perhaps the Actinomycin D in the stranded mix kills most of the SuperScript II when freeze thawed together?

So if you use a freshly made stock you might get very different results than you might get from frozen stock. My advice: mix up only enough First Strand Synthesis Mix as you will use for 1 batch of libraries. Keep the SuperScriptII in its normal high glycerol% buffer where it has high stability at -20 oC until just before use.

--
Phillip

[robertami]

Dear Phillip,

thanks for your kind reply. still can't work out what that thick band at the upper marker level is.
Illumina techsupport suggested might be genomic DNA contamination, although I did use DNAse during my RNA prep. Could it be an artifact from the bioanalyzer in your opinion?
I might run an agarose gel just to see if it's still there then.
Any suggestion is very much appreciated!
Thanks

[robertami]

Also, I ran a couple of samples for a colleague who had some problems in shearing some DNA. The BA trace shows a thick band right where the higher marker is, just like my samples.
Have you ever tried to sequence a sample that carries genomic DNA contamination?
Thanks in advance!
Roberta

[GenoMax]

Have you ever tried to sequence a sample that carries genomic DNA contamination?
Thanks in advance!
Roberta

You should expect to see something like this: http://seqanswers.com/forums/showthread.php?t=31540

[robertami]

Thanks Genomax.

That's kind of awful. I'm surprised Illumina assured me that such big product would not form clusters, thus would not be sequenced!

[pmiguel]

Dear Phillip,

thanks for your kind reply. still can't work out what that thick band at the upper marker level is.
Illumina techsupport suggested might be genomic DNA contamination, although I did use DNAse during my RNA prep. Could it be an artifact from the bioanalyzer in your opinion?
I might run an agarose gel just to see if it's still there then.
Any suggestion is very much appreciated!
Thanks

Thick band? I don't see anything other than the upper marker. There is some baseline noise making things look a little jagged, but this is common on agilent chips. Also resolution isn't great at that size, so the upper marker peak is smeared a little. Again, normal for a bioanalyzer chip.

--
Phillip

[robertami]

Ok got it. Thank you Phillip that was really helpful,

Roberta