1) Which is now the cheapest method for global quantification of human microRNAs? Microarrays, PCR arrays or RNA-Seq?
2) Which is the ideal RNA-Seq coverage for the quantification of human microRNAs? How many microRNA reads shall we expect from a certain coverage?
3) If I have my samples in two fractions, one depleted and the other one enriched for small RNAs, how should I proceed to analyze the RNA integrity? I was told that I have to analyze the integrity using the fraction depleted of small RNAs, because it contains the rRNAs used to obtain the RIN. Ok, but couldn't I use the snoRNAs present in the small RNA-enriched fraction to calculate this index? Has anyone done this?
1) Why consider indirect quantification (microarray and PCR array) when you can do directly quantify (sRNA-seq) miRNAs. A run on Miseq SE50 is can yield all the coverage you need for several multiplexed samples.
2) there is no ideal "coverage" per se. the level of coverage depends on many things, sic as 1) size of sRNA transcriptome in your specific sample, 2) sequencing platform, 3) sample quality, 4) number of multipexed samples per run. I found that 15-20 million reads per sample is quite good to capturing rare/low expressed miRNAs.
3) technically speaking, it is difficult to calculate the coverage for a small RNA experiment as we don't know the size of the small RNA (incl. miRNA) transcriptome.
4) RIN is calculated based on the 28 and 18S RNA in your sample. If you run the RNA chip, you need to use the sRNA-depleted sample. That is the Agilent made. If you want to see the sRNA integrity/distribution, you need to get the sRNA chip from Agilent.
Hope this helps.
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