I want to create RNA and miRNA libraries for the Arabidopsis root tip
and was wondering how many plants i should grow during my experiment?
According to Natalie Breakfield: It depends on the size of the root tip you are using. I hand dissected 100 Arabidopsis roots into 4 sections: meristematic zone first half (includes columella and QC), meristematic zone second half, elongation zone, and 1st 2mm of maturation zone. Each pool of 100 roots yielded about ~1 ug total RNA (extraction was done using miRVana kit from Ambion) which I then used for small RNA sequencing. I did not do any mRNA sequencing, but it should be the same.
You cant get it in single experiment, each time you take 100 mg of root tip and work. mRNA harvest depends upon the time of sacrificing the roots (active or not) and protocol perfection etc
Hallo, it is my suggestion that your sample size should be 100 mg. You should excise extreme tip portion of root tip which contain meristem zone. I hope final yield of mRNA would be at desire level, provided the proper protocol is followed.
To harvest just the root cap, meristem, elongation zone and as little maturation zone is very precise harvesting. Do you have any useful tips? Such as how many plants would you recommend per plate? More plants per plate means an easier harvest however isn't that a trade off ofr accuracy in where you the cut the root to take the sample?
ps thanks for the advice so far, i'm in the process of setting up a practice run this week...
You can ask your question from BGI company by the following emails:
You have to collect about ~85mg of root tip from Arabidopsis during half life of plants and follow spectrumTM plant total RNA isolation kit for better results.
Hello Richard,
Did you manage to answer your question by your own research?
How many root tip did you harvest at the end for RNA extraction? How old were the plants?
Thanks in advance for your help.
Bests,
Alice
Hi Alice
I used the root tips of approximate 100 seedlings to generate enough RNA for RNA sequencing (we also made a novel tool to assist with the seed sowing https://plantmethods.biomedcentral.com/articles/10.1186/1746-4811-9-41). I used 7 day old seedlings to ensure their meristem had reached its maximum size and cell elongation rates had stabilized. Let me know if you need any extra details.
Best wishes, Richard
Hello Richard,
According to your experiments, which protocol or kit is the best for RNA isolation form root tips?
Kind regards,
Mohammad
- Badges
- Science topic
- Similar topics
- Biological Science
- Botany
- Plant Biology
- Plant Biotechnology