Dealing for the first time with RNA, I'm trying to extract several samples of gram negative bacteria in order to perform a RNA-seq analysis afterwards. The problem is that I want to analyze bacteria in a very early growth phase, when OD660 is about 0.1-0.2. The experimental setup that I've tune up let me extract a total of only 5 ml, so the final yield of my RNA extraction is about 200 ng/ul (in a total of about 20 ul)
I was confident because I just need 1 ug to be treated for RNA-seq, but it seems that any extraction starting from low biomass, giving consequently a low amount of RNA extracted, is not reliable, and I should scale up my experiment to obtain at least 1ug/ul.
Thank you in advance
Depending on the library preparation protocol you use, you should be able to produce reliable and consistent libraries from
I agree with Zachary. I also produced good results with ~1ng total RNA.
I agree with both as well. I just submitted samples for RNA-seq today that passed all criteria necessary and I started with only 20,000 cells in each sample (a few hundred pg of RNA).
First 4µg of RNA is not really a small amount, that's in the northern blot scale. In general however the main problem with library preparation with small amount of starting material is loss of complexity since you run a risk that abundant transcripts would be amplified proportionally and rare ones would be lost. The more PCR cycles you do when amplifying the library the worse that problem would become. There are ways to get around that problem such as using random sequence tags (http://www.nature.com/nmeth/journal/v11/n2/full/nmeth.2772.html) but it doesn't sound like you're just there yet.
Try further diluting your adapters, in my experience that can significantly increase the quality of libraries generated from less starting material.
I agree with all that there is no "scientific reason" to have unreliable samples from few starting cells and usual less then 500 ng are Ok Which protocol are you using for the extraction? IfWhch Bacteria species are you dealing with?
I also agree with everyone, i am doing LASER CAPTURE Microdissection based RNA extraction in which the starting material is 1-2u tissue which essentially yields RNA around 50ng in total. As long as the the RNA quality is good you should go easily for downstream experiments.
Yoav said it best. Totally agree. Even if you had ultra low concentration of starting RNA you could use such kits as Clontech's SMARTer Ultra Low kit, which I have found extremely reliable.
I also agree with everyone, generally, due to the RNA are easy to degrade and the amount will be less if the transcripts of your concerned gene are less. until now we have used many kit to extract RNA such as TruSeq kit, and the results are very good, just need 500 ng to carry out the RNA-Seq(transcriptome), if you just want to quantitate the RNA in the special condition, we just need 100ng to do the downstream experiment. I am from BGI Tech (http://bgitechsolutions.com/). we provide all next generation service for global customers,
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Thank you very much for all your answers and nice advices.
I'm working with an acidophile gram - bacteria, and extracting RNA using the RiboPure RNA Purification kit, from Ambion.
Apart the possible bias in terms of low abundant transcripts than can be lost if low biomass is used to extract RNA, is it possible than the amount of total cell number to perform the extraction be in direct relationship with the purity of the RNA solution, in terms of absorbances ratio? Have any of you had experience such a phenomenon?
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