I am interested in identifying the differential expression between several genes-of-interest of different organisms.

I am trying to assess the benefits of RNA sequencing over microarrays. Both techniques are a means to an end. With both of these two methods one can "count" the differences between two conditions, e.g. cell types, time points etc. They do use different technology to achieve it, but they both give a certain number 't' in the end, which can be looked at as the strength of the expression of a specific gene under a certain cellular condition. One can than compare the two (or more) conditions to get a picture of what is different in the cell due to the changes.

As I am new to this field, can someone tell me what the advantages are of RNA sequencing over micro arrays? Alternatively, information on comparisons between microarrays and RNA sequencing methods are welcome.

  • $\begingroup$ Welcome to Biology SE! What is GOI (G of interest)? Don't assume everyone's a molecular biologist and avoid technical acronyms and technical jargon. I've slightly type-edited your question. Please remove acronyms (RNA is fine) $\endgroup$ – AliceD Mar 23 '15 at 12:25
  • $\begingroup$ No worries :) - Aha genes of interest, that makes sense. Obviously, I am not able to answer your question (otherwise I would have known) but I'm just trying to polish up your question :) Lastly, a bit more background may help you to get your question answered. I mean, sequencing and micro arrays are quite a different thing? The one is sequencing, the other one is expression profiling is it not? $\endgroup$ – AliceD Mar 23 '15 at 12:42
  • $\begingroup$ Thanks for the excellent elaboration! Adding this to the question may be a good thing (At Biology.SE many questions are closed due to a lack of underlying research effort, that's why I am bugging you :-). +1 for your question. Good luck! $\endgroup$ – AliceD Mar 23 '15 at 12:51
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    $\begingroup$ @AliceD RNA-seq is a gene expression profiling technique akin to microarrays. The sequencing step is just a technical detail. $\endgroup$ – Konrad Rudolph Mar 23 '15 at 13:37
  • $\begingroup$ Microarrays are pretty much dead. Go for a RNA-seq. $\endgroup$ – cagliari2005 Mar 23 '15 at 22:49

In RNA sequencing, total RNA is extracted, then mRNAs are purified out from the sample using polyT columns (since mRNAs have a polyA tail, this will attach to a polyT DNA chunk that is attached to some solid surface). This step is necessary because ribosomal RNAs are much more abundant than mRNAs, and for sequencing you only want to use mRNAs. Then the purified mRNA is transcribed to DNA using reverse-transcriptase and finally the cDNA is sequenced.

As for microarray a set of single stranded oligonucleotids (short DNA sequences) (aka. probes) are attached to a solid (usually glass) surface as small dots. This the so called DNA/RNA-chip. Then the chip is washed with the fluorescently labelled singlestranded sample, and the complementary strands attach to each other, the rest is washed away - this is a so called hybridization technique. The signal is then detected by reading the fluorescence signals at each spot.

The microarray technique is quite old and is less sensible and the number of probes are limited / array, while with RNA sequencing you can sequence the total mRNA population of each organism and examine anything you want. Also since most sequencing techniques include a PCR step less starting material is sufficient, and has higher sensitivity and specificity.

These wiki pages on RNA-seq and microarray technique provide detailed info on both.

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    $\begingroup$ To complete this answer, here a link to a study that compares microarray and RNA-seq. RNA-seq is definitely considered better and microarrays are less and less used. $\endgroup$ – cagliari2005 Mar 23 '15 at 22:52

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