Talk:MicroRNA sequencing

miRNA background

The article should link to miRNA but keep the background to a minimum, because the history and specifics of miRNA research are beyond the scope of this article. The drawback of having an independent section on what miRNAs are is that potentially misleading information can be introduced. For example, the article currently states that "miRNAs are the largest family of non-coding RNAs, making up greater than 1% of the human genome." Even assuming that as many as 1200 miRNAs are found in the human genome, mature miRNA sequences are coded by a fraction of 1/1000th of 1% of the haploid genome. The only way to bring the total to 1% is to call every miRNA-containing transcript a "miRNA." This is inaccurate. miRNA precursors can be found in various classes of transcript, only a subset of which are non-coding RNAs. And so forth. SpectraValor (talk) 22:21, 12 May 2012 (UTC)

I went back and looked up my references for the intro and the reference I took the 1% from, incorrectly used a previous reverence, which stated: "estimates of the num- ber of miRNA genes in the genomes of human (200–255 miRNA genes; Lim et al., 2003b), C. elegans (103–120 genes; Lim et al., 2003a; Ohler et al., 2004), and Drosoph- ila (96–124 genes; Lai et al., 2003). In each species, these numbers represent nearly 1% of the predicted genes in the genome, a fraction similar to that of other large gene families with regulatory roles, such as the homeodomain transcription-factor family."

my mistake

Reference: Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).

Ryan.rdh (talk) 12:54, 17 May 2012 (UTC)

Also, the biomarker table is incredibly useful and is a great addition to the encyclopedia. At the same time, the studies cited (at least those I looked up) did not use NGS technologies. This table would be better placed and get more views in the miRNA article or a miRNA biomarker article. This article should focus on miRNA-seq and what makes it different from other forms of RNA-seq. Otherwise, it should split up and merged into more appropriate articles. SpectraValor (talk) 22:53, 12 May 2012 (UTC)

In response to this, I put the table as merely an example of how miRNA-seq could be used to differentiate between cancers based on known combinations of miRNAs. This is also why this is not a comprehensive list as well. I did not claim or state that all these biomarks had been found with NGS techs. It is actually a mixture; for example DLBCL was done with NGS. Papers profiling miRNAs in Breast cancer and AML with NSG have also been released.

Schotte, D. et al. Discovery of new microRNAs by small RNAome deep sequencing in childhood acute lymphoblastic leukemia. Leukemia 25, 1389–1399 (2011).

Wu, Q. et al. Next-generation sequencing of microRNAs for breast cancer detection. J. Biomed. Biotechnol. 2011, 597145 (2011).

Also I think the price you quoted for qPCR is a little misleading per sample because it completely depends on the number of miRNAs being assessed. Just looking it up now an RT-qPCR kit from invitrogen (NCodeTM VILOTM miRNA cDNA Synthesis Kit and EXPRESS SYBR® GreenERTM miRNA qRT- PCR Kits) for 200 reactions costs $648 (CAN) which scaled for 384 well reaction plates can probably give about 400 reactions. This is by no means comparable to the number of miRNAs that can be assessed in a given sample in the other two methods. I realize that experiment designs come into play here but if the goal is global profiling I don't think that is an accurate number.

Ryan.rdh (talk) 12:54, 17 May 2012 (UTC)

Hi Ryan.rdh, thanks for your replies. I would point out that qPCR is routinely used for global profiling, and I think this is what the review was referring to; Life Technologies, Exiqon, and others have qPCR-based platforms that assay all or nearly all known mature miRNAs for several species for approximately the quoted price (a bit lower now, actually). While the prices from the cited review may not be up-to-the-minute, and while you or I could provide different prices from our own experience, we must try to avoid original research and continue to focus instead on verifiable sources.

I completely appreciate your work here, but I'm still not convinced that what you've written should be here, in a separate article on miRNA-seq. It seems that the only thing that differentiates miRNA-seq from other forms of RNA-seq is that the protocol usually involves prior enrichment of small RNAs. The question is, should we also have an article on lnc-RNA-seq? On SureSelect kinome-seQ? In my opinion, portions of this article would probably be read much more if they were moved to RNA-seq, miRNA (or miRNA profiling, including multiple platforms), and perhaps a biomarker article. Just my two cents. SpectraValor (talk) 21:26, 17 May 2012 (UTC)

You are correct I should focus on more variable sources. So I looked back at the article and directly under cost is listed (754 human microRNAs queried per sample), (950 microRNAs queried per sample), (theoretically, all microRNAs queried per sample). I am curious about why you omitted this? It gives proper context for those particular figures given.

Ryan.rdh (talk) 14:53, 22 May 2012 (UTC)

I'm not sure why the original table didn't include everything from the source, since I didn't add it to the article. If I remember correctly, I placed the cost data from the source into the pre-existing table. I don't think I made a conscious decision to omit anything. Although as I think about this now, I'm not sure it would add anything to the article to state how many miRNAs are queried with each of these specific platforms (Life TLDA, Exiqon hyb, etc.). In essence, each technology is capable of querying all miRNAs present in meaningful quantity. Of the hundreds queried, only about 150 to 250 will be present in any given cell or tissue type, and you will measure these same miRNAs with any platform. The remaining 500 or 700 miRNAs are just called absent. Any minor differences between platforms, as well as many supposedly "novel" miRNAs, often turn out to be bogus miRNAs, like 1274A and 1274B, or miRNAs that have extremely low abundance if they are made at all. So while I certainly wouldn't object if you wanted to place the proposed line into the table, I think the text already does an excellent job of pointing out what the real relevant strength of miRNA-seq is: finding edits, length polymorphisms, species-specific sequences, etc. SpectraValor (talk) 21:59, 22 May 2012 (UTC)

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