I'm loolikng for a RNA-seq technology tha allow to generate a MicroRNA library (total MicroRNA and not a specific screening of some of them) from less than 500 ng of total RNA extraction.
I think it's doable, but you may need to add more amplification steps in. If you can isolate your RNA using a size selection and enrich for the small RNA fraction, you should be able to get sufficient quantities of cloned miRNAs using Illumina's small RNA prep v1.5 or later.
We had low RNA amounts (10-100 ng), and even working with one of the world's best core facilities, we couldn't get it to work. I went with a 384-well qPCR format and it is working very well, even with very small amounts of RNA. Not as fancy, and not as unbiased, but it gets the job done.
Thank you so much for your answers. I would also ask you, how much RNA dou you think it's possible to extract from 1000 cells? As far as I know, not more than 20 ng....
Hello Matilde,
I have done small RNA libraries using the ilumina kit starting with 500ug of total RNA and I haven't had any problem. This protocol has a step of gel purification in which I have seen we eliminate the majority of ribosomal RNA. The only change we did was that instead of doing the 12 cycles recommeded of amplification by PCR we did 15. Besides that, in the genotranscriptomic platform of my centre do this regularly and although they recommend using as much as possible, there's no problem going even up to 250ng,
Good Luck!!
HI
we have also made small RNA libraries with as less as 100ng of RNA using illumina kit and it works well. As mentioned by Jennifer , we also do PCR amplification for 15 cycles.
Hope that helps.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA
- microRNA