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Old 01-26-2011, 11:01 AM   #1
cascoamarillo
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Default TruSeq Small RNA

Hi guys,

Has anyone used the new TruSeq SmallRNA kit from Illumina? Does it only require ONE day to complete the protocol?

Regards.
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Old 01-26-2011, 11:35 AM   #2
mnkyboy
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It can be done in a day. The rate limiting step is the gel purification and then the validation.

If you use the indexing it allows you to purify more libraries per gel which can save some time.
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Old 04-28-2011, 07:21 AM   #3
vvendr
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Default TruSeq small RNA- sample prep

Hi,

has anyone ever tried to use small RNA as starting material? What small RNA concentration is suggest?

Thanks
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Old 04-28-2011, 07:33 AM   #4
cascoamarillo
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Quote:
Originally Posted by vvendr View Post
Hi,

has anyone ever tried to use small RNA as starting material? What small RNA concentration is suggest?

Thanks
Hi,

I've been using only small RNA fraction as starting material; but not with small RNA kits (truseq, neb,...), I did it with a more traditional approach. I did get better results than with total RNA as a starting material. The amount of small RNA?... I'm not sure; I just tried to obtain the 20 ug of total RNA (that most of the kits recommends) from the extraction and then isolated only the fraction that I needed.
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Old 04-29-2011, 06:04 AM   #5
vvendr
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Hi,

I was actually wondering how mauch smallRNA I need to start, if I have smallRNA extracted without previous totalRNA isolation.
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Old 02-09-2012, 12:31 AM   #6
sterakura
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Hi,
Have you seen artifact sequences "CCACGTTCCCGTGG" in TruSeq Small RNA system?
I often found this sequence in TruSeq SmRNA system, not in old Small RNA System (v1 & v1.5).
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Old 02-09-2012, 08:50 AM   #7
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A protocol I am testing says 1 to 10 ng of small RNA or 1 ug of total RNA.
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Old 02-10-2012, 09:01 AM   #8
vvendr
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Finally, I asked directly to Illumina tech-support, and the answer was 20-50 ng. So, I used that siRNA initial amount and it worked!
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Old 02-20-2012, 07:37 PM   #9
CC_seqanswers
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Could you elaborate a bit on how the results with small RNA are better than with total RNA?Thanks.

Quote:
Originally Posted by cascoamarillo View Post
Hi,

I've been using only small RNA fraction as starting material; but not with small RNA kits (truseq, neb,...), I did it with a more traditional approach. I did get better results than with total RNA as a starting material. The amount of small RNA?... I'm not sure; I just tried to obtain the 20 ug of total RNA (that most of the kits recommends) from the extraction and then isolated only the fraction that I needed.
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Old 02-21-2012, 06:57 AM   #10
cascoamarillo
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Quote:
Originally Posted by CC_seqanswers View Post
Could you elaborate a bit on how the results with small RNA are better than with total RNA?Thanks.
Hi,

MY results were better with the small RNA fractionated sampled than with total. How? I'm not sure; the only thing I know is at the final step, the PCR amplification, I obtained better and clear band (around 100bp) with the size selected material.
Best
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Old 05-01-2012, 07:35 AM   #11
chaos81
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could you elaborate on the more traditional approach you took to getting your small rna fraction? All our preps have come from total rna and we never get a defined peak in the size select area, we always get a smear unfortunately. thanks
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Old 05-01-2012, 12:46 PM   #12
cascoamarillo
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Quote:
Originally Posted by chaos81 View Post
could you elaborate on the more traditional approach you took to getting your small rna fraction? All our preps have come from total rna and we never get a defined peak in the size select area, we always get a smear unfortunately. thanks
What I did was to size select the RNA fraction: 15-20% PAGE in denaturing contitions, cut the gel band (15-25 or 20 -35; it depends what it's for); elute ON in salt solution and precipitate. And the size selected RNA is used for the library construction (3' ad ligation, etc...). Hope it helps.
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Old 05-03-2012, 07:59 AM   #13
seqfan2011
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Has anyone tried Truseq small RNA kit with <100ng total RNA input? We have some precious samples for microRNA study, but can't seem to find any kit on the market supporting such low input. Any thoughts?

Thanks
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Old 06-28-2012, 12:32 PM   #14
Elizabeth
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Quote:
Originally Posted by sterakura View Post
Hi,
Have you seen artifact sequences "CCACGTTCCCGTGG" in TruSeq Small RNA system?
I often found this sequence in TruSeq SmRNA system, not in old Small RNA System (v1 & v1.5).
Did you ever find out what this is? I am seeing it too in my TruSeq smRNA libraries.
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Old 09-17-2012, 02:05 AM   #15
vtosha
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I think when small RNA is used instead of total RNA then reagents are used more economically and with more good. And clear bands at the final step too.
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Old 09-20-2012, 03:40 AM   #16
pmiguel
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Quote:
Originally Posted by sterakura View Post
Hi,
Have you seen artifact sequences "CCACGTTCCCGTGG" in TruSeq Small RNA system?
I often found this sequence in TruSeq SmRNA system, not in old Small RNA System (v1 & v1.5).
Quote:
Originally Posted by Elizabeth View Post
Did you ever find out what this is? I am seeing it too in my TruSeq smRNA libraries.
I don't know what it does, but:

It matches the "Stop Oligo (STP)" sequence in the Illumina Customer Sequence Letter. "STP" is the three letter code for "Stop Solution" in the TruSeq Small RNA prep kit. Added as the last part of the T4 RNA Ligase2 (deletion mutant) ligation of the 3' adapter to the RNA. The sequence also has the footnote "patent pending" in "the Letter". Not sure what patent that would be.

--
Phillip
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Old 09-20-2012, 08:04 AM   #17
vtosha
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NEB kit use heat inactivation of ligase (see instruction to ligase). As I think Illumina use NEB-reagents for it's sample prep kits, so I try to change Illumina manual and use temperature inactivation instead Stop Solution and I had normal libraries.
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Old 04-25-2013, 01:02 AM   #18
beibeizhou
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Default truseq small RNA library

how many library yield can you get using truseq small RNA kit ?
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Old 05-23-2013, 01:40 AM   #19
vtosha
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1-20 nM. We use a half dose of reagents.
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Old 10-01-2013, 12:01 PM   #20
katjef
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We have ribosomal RNA-depleted samples that we want to examine both long RNA (mRNA, lncRNA) as well as small RNAs. Do we need to prepare 2 separate libraries for small and long RNA? Or would a Truseq (Illumina) library prep be sufficient to get all RNA types?
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