Best Library kit for illumina low amount RNA

[ildem]

Hi all,
we are interested in doing RNA-seq (for differential gene expression analysis) on total RNA samples. Our sequencer is illumina HiSeq 2500.
We have approximately 20-50ng of RNA per sample.
I have found the following kits for library preparation:

1. Takara/Clontech
SMARTer Stranded RNA-Seq Kits—Strand-Specific Library Construction for Transcriptome Analysis on Illumina Platforms
needs 100 pico to 100 ng

2. TruSeq kits seem to need a min 100 ng.(right?)

3. NEBNext Ultra RNA Library Prep Kit for Illumina
Total RNA:
10 ng – 1 μg

4. Epicentre
ScriptSeq™ v2 RNA-Seq Library Preparation Kit
0.5ng-50 ng

5. Nugene Ovation® RNA-Seq System V2
min 0.5ng RNA


We heard NEBNext is good, and we have used Nugene(worked ok)

In your experience, which is the best for gene expression analysis?

Many thanks!

[RNA]

Need a little more information to make a clear recommendation

Hi ildem,

Before we help you decide which kit is best, it would be nice to know more about the quality of the RNA you will be using in this study. Is it high quality RNA from fresh samples? Or is it degraded, or even from FFPE samples?

Also when you say you want to do "RNA-Seq on total RNA" samples do you mean Total RNA-Seq (using RiboZero) or just that you want to start with Total RNA and do a polyA capture based mRNA prep?

Are you interested in mostly coding mRNA or do you also want to look at non-coding RNAs, many of which may not have a polyA tail?

Regardless of these answers 20 to 50 ng of total RNA is not really that low these days. Many researchers successfully use the TruSeq kits from Illumina at these levels, even though they are spec'd out for 100 ng.

If you have good RNA (RIN of >8), and want to study differential gene expression levels, then I would try the TruSeq Stranded mRNA Sample Prep Kit from Illumina.

Good Luck!

Sincerely,

RNA

[ildem]

Dear RNA,
Thank you very much!

We have good RINs (9-10) just not much of the RNA and its quite precious.
Would you then use the true-seq kits?

If we were to have lower amounts, say5-10 ng, with good RINs, which would you then recommend?

many thanks!
i

[ildem]

Dear RNA,
Thank you very much!

We have good RINs (9-10) just not much of the RNA and its quite precious.
Would you then use the true-seq kits?

If we were to have lower amounts, say5-10 ng, with good RINs, which would you then recommend?

many thanks!
i