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Old 01-11-2012, 08:45 AM   #1
Oliviervg
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Location: Brussels

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Question Scriptseq V2 : fragmentation problem ?

Hi,

We want to study the transcriptome of breast cancer cell lines and tissue, so we want to do RNA-seq experiment.
We prepare our sample with Tripure extraction, clean up (Qiagen), DNAse treatment. We check the quality with the Bioanalyser, our samples have a RIN of about 8 - 8,5.
Then we use the Ribozero kit with 2g of total RNA as input. We use the clean and concentrator (Zymo) to clean our sample. We obtain about 12ng of ribodepleted RNA (0,6ng/l in 20l). We check our samples with the Bioanalyser (see http://www.fichier-pdf.fr/2012/01/11...bozero-090112/), everything seems ok.
Then we prepare the library (scriptseq V2) with 9,5 l of our ribodepleted RNA, we exactly follow the protocol (We purify the cDNA with the MinElute PCR purification kit in step 4.D., and we purify the RNA-seq library using AMPure XP purification (step 4.F.) , and we perform 15 cycles for the PCR)
After that, we quantify with the Qubit, and we obtain +/- 3 to 5 ng/l of DNA (total amount 60 to 100 ng, it's less than expected).

When we put 1l of our sample on the Bioanalyser (DNA 1000), we see nothing, no peak, no concentration.

We tried one qPCR with this DNA (+/- 5ng DNA per well) , and we obtain good Cq values (we tried Actin (Cq = 17,2), 18S (Cq = 25,5), 28S (Cq = 22,1).

So we suppose that the fragmentation step didn't work well.
We did all the experiment twice, and we obtain similar result.

What do you think about that ?
Has someone else use this kit ? If yes, do you have any problem ?

Thank you

Best regards,

Last edited by Oliviervg; 01-11-2012 at 08:50 AM.
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Old 01-11-2012, 08:56 AM   #2
huguesparri
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I'm not familiar with the scriptseq V2 so what I'm going to say might be silly but did you try to run your library on an Agilent HiSensitivity Chip?
Maybe your concentration is too low to be detected on DNA1000 Chips...
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Old 01-12-2012, 02:03 AM   #3
Oliviervg
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Thank you for your answer,

After the library preparation we get more than 3 ng cDNA / l, that is sufficient for an analysis with the DNA 1000 on the BioAnalyser.

We 'll try an other control on a DNA 7000 chip, and hope to see something.

Does someone already use this kit ? Did you have problem ?
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Old 04-25-2013, 12:45 AM   #4
susma
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Hi,
I am using this kit now. It was my first time, but my RNA source is bacterial. I put 40 ng rRNA depleted mRNA as input and I got 30 ng/ul (Total volume 20 ul) DNA at the end, then I run an Agilent HiSensitivity Chip but I got strange results with the fragmentation. I got many fragments!

I am trying now to ask them what might be wrong.
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