TruSeq Small RNA

[cascoamarillo]

Hi guys,

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

Regards.

[mnkyboy]

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.

[vvendr]

TruSeq small RNA- sample prep

Hi,

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

Thanks

[cascoamarillo]

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.

[vvendr]

Hi,

I was actually wondering how mauch smallRNA I need to start, if I have smallRNA extracted without previous totalRNA isolation.

[sterakura]

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).

[NextGenSeq]

A protocol I am testing says 1 to 10 ng of small RNA or 1 ug of total RNA.

[vvendr]

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!

[CC_seqanswers]

Could you elaborate a bit on how the results with small RNA are better than with total RNA？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.

[cascoamarillo]

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

[chaos81]

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

[cascoamarillo]

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.

[seqfan2011]

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

[Elizabeth]

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.

[vtosha]

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.