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Old 09-26-2011, 11:35 AM   #1
OnUrMark
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Default TruSeq RNA initial concentration and Ampure beads

Greetings!

I am getting ready to use the TruSeq RNA kit for the first time. I noticed the concentration of Ampure beads to sample decreases as you progress through the steps to prepare an RNASeq sample. I assume that as the bead concentration to DNA decreases, the maximum length of fragment washed away will increase.

Ex: 1.8:1 bead: DNA removes anything less than 100bp. Then perhaps 1:1 bead: DNA would remove anything less than 200bp.

1) Is this a correct assumption?
2) Has anyone found that varying the initial RNA concentration affects the maximum length of fragment washed away and results in a different final fragment length?

Thanks in advance for your help and sharing your knowledge on this preparation technique.
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Old 09-27-2011, 04:31 AM   #2
RNAseqer
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According to what I've read anything greater than a 1x ratio will recover everything <~100. Its only when you go less than 1x do you start being able to size select, ie. .5X or .75X. I'd be interested in the experience of others too.
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Old 09-27-2011, 08:29 AM   #3
OnUrMark
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Hi RNAseqer,

I am guessing you mean a 1x ratio will recover everything >~100bp. I am concerned if this is true because adapter dimers must be removed, and they are ~120bp.

Thanks,

OnUrMark
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Old 09-27-2011, 08:37 AM   #4
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In general (not speaking specifically about the TruSeq kit, and its adapters)...but 1x will definitely recover a large fraction of 120bp dsDNA. Even very aggressive cuts (0.5x) will leave a lot of 100bp fragments (by gel staining and PCR).
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