I am planning to study the miRNomes in different diseases. Does anyone have experience with the analysis of miRNomes using NGS, especially when starting with a low amount (2-10 ng) of enriched miRNA? Any kind of information, especially in the generation of sequencing libraries (with or without amplification) would be helpful for me. Thanks for your efforts! Best, CK
I am interested in doing the same thing, I have read that the Epicentre kit can work with low input amounts of enriched miRNAs. Also, you can try with the Truseq kit, but what you do is you ligate the adapters and then do a qPCR so that you can see at which cycle number you are within the amplification stage and then use that amount of cycles for the PCR when creating your library. Best of luck.
I am interested in doing the same thing, I have read that the Epicentre kit can work with low input amounts of enriched miRNAs. Also, you can try with the Truseq kit, but what you do is you ligate the adapters and then do a qPCR so that you can see at which cycle number you are within the amplification stage and then use that amount of cycles for the PCR when creating your library. Best of luck.
Hi Stefanie, thank you for your fast reply. I already worked with Epicentre kits for the amplification of sRNA and can highly recommend them. I did not know that they also provide a kit for miRNA amplification, but I will try it!
It is the same kit, the small RNA kit. Types of Small RNAs Included in the ScriptMiner Library: RNA 3′-end modifications: The standard kit reaction produces a library from RNAs with 3′ ends containing either a 2′,3′-OH group (such as mammalian miRNA) or a 2′-O-Me,3′-OH group (such as piwi-interacting RNA [piRNA] and plant miRNA).
Our lab compared TruSeq and NEBNext and had better results with NEBNext. We started with a total RNA sample (miRNeasy), added adapters, and did 15 rounds of PCR amplification. To separate microRNAs from larger RNA species, we used Pippin Prep which usually gives us about 70% yield.
I intend to study miRNa in cardiovascular disease using MiSEQ from illumina, using sample collection from BD Paxgene, Do you pleople think it is a good strategy to enrich miRNA using these same Pippin Prep system? and Miseq system from illunima?
thanks
mario
In my view, we can study the miRnome with both NGS and microarrays. In my lab, we have used successfully oligoarrays Agilent to study the expression of microRNAs. These arrays are sensitive enough to start from nanograms of total RNA. The methods of data analysis are also appropriate (platform R). But if you really need to know if there is a new microRNA involved in your model-system, you'll have to sequencing and re-sequencing their RNAs.
Can you send your e mail Geraldo, where unity are you?
I apreciate if you contact me to discuss our idea
mario
Hi Mario, I am at Department of Genetics, Faculty of Medicine of Ribeirao Preto (USP).
Emily, we used the 3% Pippin Prep gel and excised a band between 120 and 160 bp. Our adapters were 120bp, so we were essentially taking RNA less than 40bp. If you are interested in looking at both mature and pre-microRNAs, I would recommend taking a band between 120bp and 300bp. For sequencing, we have had success with both Illumina HiSeq and MiSeq. (MiSeq is more suitable for smaller mirNomes like from exosomes. Hope this helps.
Dear Christopher, can you please tell me which company sell Pippin Prep gel?
thaks
mario
Dear Christopher, I appreciate you quickly anwer. I think there is no representative in Brazil. Thanks
mario
Hi Mario, just coming back to your question regarding if MiSeq is a good system for you to use, it also depends on how many samples you have; if you only have a few then MiSeq would be fine, but if you have say 10 or more, then it would perhaps be more cost effective to do it on the HiScan.
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