I would use the advice on which technique to choose for lncRNA profiling.
I am interested in comparison of the preparation time and data analysis/price/quality between RNAseq or microarray. I am looking forward to some tips.
I agree with that advice. The value of true biological replicates can out weight the use of an NGS platform if resources are limited. Be clear about the question you are asking and determine which approach will give you the answer you need with the most confidence. Among other advantages, NGS can give you insight into the diversity or your samples, microarray can give you statistical power to distinguish noise from true signal.
RNA-Seq is certainly better way to find out novel transcripts or lncRNA, which can not be captured on micro arrays. however, if you are analyzing relative expression of some known lncRNA, then micro-array is fine..I agree with Rohan, adequate biological replicates and costs need to be the considered for planning the experiments. But most likely current research focus has been to explore novel lncRNAa and follow through the characterization, the field is really picking up...
Best wishes!
Very good question. No simple answer. We use both a custom microarray (containing all human mRNAs and 17,000 lncRNAs) and RNA sequencing for lncRNA gene expression analysis, depending on the study goals and budget (see attached link for the technologies and services we provide at Biogazelle, a Ghent University spin-off). Importantly, when using sequencing for lncRNAs, we recommend rRNA depletion (instead of polyA selection), directional sequencing (preserving strand information), and last but not least sufficient read depth. 50 million paired reads are really needed for tissues, as there are so many and low abundant lncRNAs.
http://www.biogazelle.com/lab-services/carefully-selected-analytical-platforms
If you are interested in de novo discovery, I'd recommend RNA-seq approach. Microarrays in principle only allow you for profiling of already known RNAs.
RNA-seq period. The prices are crashing down. Do total RNA with ribosome depletion and at least 20 million reads per replicate. No need for more than 3 replicates (biological that is)
Here are the challenges of lncRNA differential analysis by RNA-seq:
· For mere detection of the presence of an lncRNA, a few reproducible counts unique to that lncRNA are required.
· But for quantification, at least hundreds read counts are required. LncRNAs as a population are ~10x less than mRNA. Count numbers at these levels are not nearly sufficient for differential expression analysis.
· Many lncRNAs have complex overlapping relationship with mRNA genes, sequencing reads from an lncRNA are not necessarily unique to the lncRNA, which demands deep sequencing reads to construct transcripts for novel lncRNA discovery.
In short, a large proportion of lncRNAs are not sufficiently quantifiable by RNA-seq at regular sequencing depth. Deep, paired-end, strand-directional sequencing is required for quantification and novel lncRNA discovery.
I think this question is still relevant as the field is getting mature to address the biology of several lncRNAs.
The link bellow from Arraystar gives a better comparison of both technologies
http://www.arraystar.com/reviews/arraystar-lncrna-microarrays-better-than-rna-seq-for-lncrna-expression-profiling/
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