Ultra Low Input mRNA‐Seq

[Simon J Foster]

Has anyone tried Illumina's recommended protocol for Ultra Low Input mRNA‐Seq using the Clontech SMARTer Utra Low RNA kit?

If so, any comments on success (or not) would be very welcome. Also if anyone has any alternative techniques? One of our researchers wants to isolate and sequence RNA from haustoria which are small projections of fungal/oomycete hyphae within host plant cells.

Thanks.

[NextGenSeq]

I haven't tried it but one thing I don't like is that they don't support barcoding. With the HiSeq you'll get plenty of coverage to multiplex with RNA-Seq

The NuGen Ovation RNA-Seq system supports barcoding and you can start with 500pg or less.

[ScottC]

Can't you just use it and then ligate multiplexing adapters?

[monad]

DSN works for sub pg level of input RNA (not detectable with Qubit). total RNA was from single cell layer of insect brain and impossible to secure enough total RNA for 500pg range. We got 30M passing reads from GAIIx lane and turn out to have 5M unique reads! This is amazing output from little input material.

[greigite]

Can't you just use it and then ligate multiplexing adapters?

We did that with the Nugen Ovation kit and got great results (generated cDNA with Nugen kit from small amount of input RNA, then ligated inline barcoded adapters). The only drawback of Nugen is cost, ~$2K for 8 reactions.

[kwaraska]

We have used the Ovation Kit.

The one thing I will warn you is to do all work in a PCR workstation or equivalent. Take your pipettes apart and clean them too.

Any little bit of contamination will amplify as well, throwing off everything.

Can you tell---It happened to us!

[seqfan]

Trying to refresh this thread. Does anybody have experience with the Ultra Low RNA Kit for Illumina Sequencing (http://www.clontech.com/products/det...6487&tabno=1)? Also, has anybody compared Nugen's Ovation kit (http://www.nugeninc.com/nugen/index....seq-system-v2/) to the Ultra Low RNA Kit for Illumina Sequencing?

[zianeffy]

Excuse my ignorance, but what is the "DSN" that monad was talking about?
Thanks

[protist]

Excuse my ignorance, but what is the "DSN" that monad was talking about?
Thanks

DSN is double stranded nuclease - the protocol allows normalization of a library ultilising the enzyme to digest out the overabundant species eg rRNA

Overview PDF and Illumina protocol attached.

[454newbie]

Has anyone had any experience using the SMARTer ULTRA followed by Nextera?

[pie]

ONLY 1UL start quantity for Clontech SMARTer ultra low RNA kit

how have users got around having to use just 1uL of RNA for this kit?

i am extracting minute amounts and obtain them in 12uL. have people been etoh precipitating these amounts and blindly resuspending in 1uL? it seems problematic to me.

[at2253]

Does anyone know how SMARTer II A Oligonucleotide (Clontech) and TTO (Epicentre) from low input kit work? And is there anyway to replace them or figure out the sequence, the kits are very expensive?

[Him26]

If you do correlations between amplified and unamplified what would you get for R^2 values?
And how are you isolating single cells or ultra low RNAs?

[canted]

DSN works for sub pg level of input RNA (not detectable with Qubit). total RNA was from single cell layer of insect brain and impossible to secure enough total RNA for 500pg range. We got 30M passing reads from GAIIx lane and turn out to have 5M unique reads! This is amazing output from little input material.

Has this been published? Does anyone know of published RNA-seq studies using the SMARter ultra low RNA kit?

[weigrc]

Last month, I've asked Clontech about the publication using SMARTer kit. They replied since it was launched on 31th-Mar-2011, no publication by now.