TruSeq RNA initial concentration and Ampure beads

OnUrMark · 09-26-2011, 12:35 PM

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.

RNAseqer · 09-27-2011, 05:31 AM

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.

OnUrMark · 09-27-2011, 09:29 AM

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

ECO · 09-27-2011, 09:37 AM

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).

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
AMPure XP vs SPRI beads	Tali7	Sample Prep / Library Generation	19	07-12-2017 12:13 PM
AMPure, amplicons and GS Jr enrichment beads	suefo	454 Pyrosequencing	17	02-06-2012 04:24 AM
concentration of TruSeq adapter stocks?	jlove	Illumina/Solexa	9	01-15-2012 09:20 AM
TruSeq DNA adapters in RNA-seq prep... concentration?	ScottC	Sample Prep / Library Generation	4	09-10-2011 03:38 AM
initial amount of total RNA or enriched small rna for small rna seq	Deli Çoban	Sample Prep / Library Generation	4	06-04-2011 04:47 PM

09-26-2011, 12:35 PM	#1
OnUrMark Member Location: Oregon Join Date: Jan 2011 Posts: 24	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.

09-27-2011, 05:31 AM	#2
RNAseqer Member Location: London Join Date: Sep 2010 Posts: 22	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.

09-27-2011, 09:29 AM	#3
OnUrMark Member Location: Oregon Join Date: Jan 2011 Posts: 24	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

09-27-2011, 09:37 AM	#4
ECO --Site Admin-- Location: SF Bay Area, CA, USA Join Date: Oct 2007 Posts: 1,358	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).