Depends on how many genes you want to measure. You can do RNA-seq with a very low quantity of RNA but if you aren't interested in the entire expression profile it would be better to design a custom targetseq or to run QRT-PCR.

Why not use a simple and standard sequencing technique (like fluorescence-based ddNTP capillary sequencing) if you have one or two genes? To sequence the whole genome when you are interested in one or a couple of genes seems a waste of money. qPCR may be an alternative if you are interested in gene expression only. But it will not give you sequence information.

You would be checking expression level following target enrichment??? I doubt, whether it is at all a feasible option. Rather I appreciate the suggestion made by Jennifer. There are lot of issues, which are apparently not there, but one will face while doing RNA seq. To start with you need to know what is the size of the genome? If genome size is small, then one can easily accommodate the transcript into a smaller capacity run too, like 454 (Still I am not sure about it) But yes, if you are dealing with Human sample, you need to ribosomal RNA depletion. For that, you need to have at least 4-5 micro gram of total RNA (DNAae treated pure) RNA for smooth proceeding. FYI.. presently in one Illumina Flow cell you can accomodate 24 Total human Transcript with ~100 million reads each.

Targeted RNA-Seq is certainly possible, but it is really best suited for looking at rare transcripts and discovering novel splice isoforms. It will almost certainly skew the relative expression levels, probably to a very large extent, so I wouldn't recommend it for expression profiling. Exactly how much is a "very small amount of material"? Standard inputs are 100ng total RNA (assuming the quality is high), but good results have been achieved with 1 ng of total RNA with the standard protocols. Using the SMART kit from Clontech, you should be able to get down in the sub-nanogram range.

Or, if you are truly only interested in a subset of genes, you could use qPCR, dPCR or maybe even Nanostring.

Thank you for all your suggestions. Sorry if I haven't state my questions clearly - we are interested to know the expression level of only a small number of human genes, out of human tumor tissue sample. But the amount of sample is much less than what usually required by a standard RNAseq study, so I'm considering whether do target enrichment of only these several genes and then run RNAseq, or just QRT-PCR as Jennifer suggested. As for the amount of RNA - we are not sure now since we are just planning the study, but since the tumor is very small out of bone marrow, a rough estimate may be as low as 10-20ng or even less. This is OK for RT-PCR but I'm not sure whether and how it works for RNAseq.

Xin, target enrichment followed by RNA-Seq is not a good solution for you. You'll destroy the relative expression levels through the normalization effect of target selection. Deciding between qPCR and RNA-Seq (yes, even with the small amounts you have) probably depends on two things:

1) How intact (good quality) is the RNA? If it is in good shape, 10-20ng should produce informative and reproducible expression profiles with standard RNA-Seq protocols (although you could also easily switch to the low input protocols).

2) What are the expected expression levels of the genes in question? If they are very low expressers, the low input RNA-Seq may not be the best choice as these are the first genes to drop off.

If you have good quality RNA and aren't looking for genes with very low expression levels, I'd go with RNA-Seq. You'll get more information that might be useful down the road.

If you truly know that you only want info from that handful of genes and/or your budget is very constrained, qPCR might be the better option.

Thank you Shawn for the good suggestions, I'm also worrying that the target enrichment will introduce additional bias and variation to the measurement of RNA level. I think I will try to see if there are public data in the database for relative expression level of my target genes then to decide if RNAseq has any chance of success. But thank you everybody for the very helpful discussion.

still i am not convinced with the idea of targeted RNA seq. if you also want to know expression. Please provide me some literature on this

Maybe you check out this: http://www.rna-seqblog.com/methods/library-preparation-methods/small-rna-sequencing-library-preparation-20/

Single cell transcriptomics is almost impossible as you need to be very accurate in all steps of the experiment and the dynamics of transcript generation and degradation are hard to rule out.

Just got the link for newer and cheaper library prep methodology there... Please provide me some link on targeted RNA seq protocol.(also informative for expression study)