Small amount of RNA to start with?

[answersseq]

hello,
I am looking to check expression with very limited amount of RNA. Has anyone ever tried to see the lower limitation for making cDNA or label miRs?
Thanks!

[manoj.b]

Small amounts of RNA as starting material.

Hi ,

You can even amplify severely degraded RNA and small amounts of start up material in the range of (50 pg - 500 ng) of total RNA and the yield from ampification would be in the range of 6 - 10 ug.

Such kits are available with few companies like Ocimum Biosolutions and you can mail your query to [email protected]

MJ

[Ant_J]

Hi,

We have successfully used RNA from laser microdissected cells (2 ng-20 ng range of total RNA) to produce dsDNA for 454 sequencing with good results. The initial cDNA fragment size was ~300 bp for a final read size of 220 bp (two round of RNA amplification).

Also we used one round of amplification for a cDNA fragment size around 700 bp starting with fresh (pollen) RNA.

Check EPICENTRE Biotechnologies for their different kits (http://www.epibio.com/category.asp?CatID=74).

(Note: I'm not associated with this company).

[timread]

Can anyone direct me to any published literature that started with very small amounts of RNA (10-100pg), amplified and sequenced on Illumina? thanks in advance

[Ant_J]

Don't know with Illumina, but in this one used 454 sequencing starting with very small quantities of RNA.

Global gene expression analysis of the shoot apical meristem of maize (Zea mays L.).
Ohtsu K, Smith MB, Emrich SJ, Borsuk LA, Zhou R, Chen T, Zhang X, Timmermans MC, Beck J, Buckner B, Janick-Buckner D, Nettleton D, Scanlon MJ, Schnable PS.
The Plant Journal
Volume 52 Issue 3, Pages 391 - 404

http://dx.doi.org/10.1111/j.1365-313X.2007.03244.x

They used a protocol based in Ambion products that it seems to work great with maize samples but that didn't work for tomato fruits tissues for us. We are now with the assembly of 454 samples from amplified RNA using Epicentre kits.

[answersseq]

haha, I guess we are on the same boat. I actually can only get ~300pg from one preparation..

Can anyone direct me to any published literature that started with very small amounts of RNA (10-100pg), amplified and sequenced on Illumina? thanks in advance

[manoj.b]

Protocol for low amount of RNA

sorry for the delayed reply

The following is the link for ExpressArt® mRNA amplification Pico kit is
suitable for extremely low amounts of less than 1 ng of input
total RNA
http://www.amp-tec.com/assets/downlo...20protocol.pdf.

here are some publication for your reference :

http://www.amp-tec.com/assets/downloads/Campean%20et%20al%202008.pdf

http://www.amp-tec.com/assets/downloads/Su%20et%20al%202008.pdf

http://www.amp-tec.com/assets/downloads/Su%20et%20al%202008.pdf

http://www.amp-tec.com/assets/downloads/Su%20et%20al%202008.pdf

http://www.amp-tec.com/assets/downloads/Su%20et%20al%202008.pdf

[SharmaM]

Hello everyone. New to NGS. I have run small RNA library on Miseq using Truseq small rna library preparation kit from Illumina. Got very low clusters 153 k/mm2. the final library concentration as measured with qubit ranged from 5-20nM & final used library was 10pM. Could anyone suggest the plausible reason for the same as well as what need to be done for increasing the cluster density.