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Old 05-25-2012, 01:56 PM   #1
gendxdoc
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Default Low input micro RNA libraries

I would like to generate small RNA/miRNA libraries from small quantities of total RNA. The Illumina TruSeq Small RNA Library Prep kit requires an input of 1ug of total RNA for input.

Has anyone tried to find the lowest input required -- while still maintaining the appropriate library complexity ? Ideally, I'd like to start with 1-50ng of total RNA.

Is there an alternate low input small RNA protocol that anyone's aware of ?
Any help would be greatly appreciated !
Thanks,
Michael
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Old 05-31-2012, 08:00 AM   #2
NextGenSeq
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miRNA libraries are the most difficult libraries to make, in my opinion. We find we get better libraries starting with miRvana purified small RNA. Libraries made with total RNA get a lot of other junk in them.
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Old 06-11-2012, 08:11 AM   #3
mikaelk
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I can only agree with this. smallRNA libraries are difficult to do and I do not recomment starting with totRNA.
In my experience (but I don't know the organism you are working with), the advised 1g of totRNA tends to be quite short, we have even increased to 10ug in certain conditions to be able to get a library.
The best and cleaner way we found out is to start with a denaturing page size selection. It is long but at least, you know exactly what you are putting into ligation (always checking with an agilent small rna chip).
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Old 07-27-2012, 05:29 AM   #4
smalan
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Hi
I have extracted small RNAs and would like to sequence the miRNAs with the Illumina TruSeq kit. However, I have very low concentrations though (around 4ng). Do you have any advice/literature that could help me?
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Old 07-27-2012, 05:57 AM   #5
mikaelk
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Hi,

Well, it depends a little on what you exactely intend to do on what organism.
I have get succesfull libraries with the protocol without any modification starting with 7L of 12000pM (so 84 nmol) sample.
You can even use lower amounts if you include a 5' decapping step prior to any ligation. For example in my organism, the 5' tends to be tri-phoso, but with a TAP treatment, we do get significantly more material from the same starting material. It has worked with 9nmol.

I do not know if those are my lower limit.

Then you might certainly do not get a lib of 2nM or more (even with a precipitation step), you have then to adapt a bit the denaturation protocol.

But again, everything is quite organism/prep modification dependant.

Hope this helps a bit.
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Old 08-01-2012, 06:06 AM   #6
smalan
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Thanks mikaelk

The miRNA were extracted from rat brain samples. We will include the 5' capping step yes and sequence it on the Illumina HiScan SQ for differential miRNA expression analysis. So you have also used the Truseq kit with your samples with low concentrations and it worked fine, right?
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Old 08-01-2012, 06:09 AM   #7
smalan
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At NextGenSeq: what kit do you use to sequence your miRNAs?
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Old 08-01-2012, 06:18 AM   #8
mikaelk
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Quote:
Originally Posted by smalan View Post
Thanks mikaelk

The miRNA were extracted from rat brain samples. We will include the 5' capping step yes and sequence it on the Illumina HiScan SQ for differential miRNA expression analysis. So you have also used the Truseq kit with your samples with low concentrations and it worked fine, right?
Yes with the amounts mentioned above.
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Old 08-20-2014, 03:04 AM   #9
CuriousA
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Hey,
Has somebody experience with this Kit from SeqMatic:
http://www.seqmatic.com/products/mirna/

It claims to be working with as little as 10ng of total RNA.

Best,
Anja
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Old 09-03-2014, 07:02 AM   #10
tchiang
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Hi Anja,
I have tried SeqMatic's miRNA kits a few times and produced successful libraries. I have used both versions of the kit and it seems that the version 2 kit produces fewer adapter dimers and better results. I haven't tried using as little as 10ng of total RNA but I am using about 100-200 ng of total RNA from FFPE samples.
I am still fairly new to NGS and the people at SeqMatic have been very helpful with questions I have.
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Old 09-04-2014, 05:09 AM   #11
CuriousA
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Thanks tchiang for your input =)

I think I'll give it a try with the v2 kit.
I also already experienced very helpful seqmatic staff answering questions!
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Old 04-25-2015, 08:44 AM   #12
rmash
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Quote:
Originally Posted by mikaelk View Post
Hi,

Well, it depends a little on what you exactely intend to do on what organism.
I have get succesfull libraries with the protocol without any modification starting with 7L of 12000pM (so 84 nmol) sample.
You can even use lower amounts if you include a 5' decapping step prior to any ligation. For example in my organism, the 5' tends to be tri-phoso, but with a TAP treatment, we do get significantly more material from the same starting material. It has worked with 9nmol.

I do not know if those are my lower limit.

Then you might certainly do not get a lib of 2nM or more (even with a precipitation step), you have then to adapt a bit the denaturation protocol.

But again, everything is quite organism/prep modification dependant.

Hope this helps a bit.
What is the protocol for 5' decapping?

What is the reason for this step?

Thank you!
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