Hello everyone, currently, I'm trying to do RNA extraction from seeds of Arabidopsis. I'm using the protocol below:
Article RNA isolation from siliques, dry seeds, and other tissues of...
I use about 90 mg of Arabidopsis seeds for extraction. What I got from the extraction is RNA with a concentration of 74.5 ng/uL and an A260/A280 ratio of 1.9 (I checked it by using Nanodrop). To dissolve the pellet, I add 50 uL of RNAse free water. Is this quantity worrisome, and should I do RT-PCR with this concentration?
If the concentration is 74.5ng/uL I would say that is okay. I would suggest running about 1ug on a gel to see if you have any degredation. Yes with that concentration you could do RT-PCR. For instance with SuperScript II from Thermo Fisher it can do quantities of total RNA from 1ng to 5ug. So you should easily be able to use what you have for RT-PCR. The only other thing I would worry about is the degredation.
If the concentration is 74.5ng/uL I would say that is okay. I would suggest running about 1ug on a gel to see if you have any degredation. Yes with that concentration you could do RT-PCR. For instance with SuperScript II from Thermo Fisher it can do quantities of total RNA from 1ng to 5ug. So you should easily be able to use what you have for RT-PCR. The only other thing I would worry about is the degredation.
Various factors may effect the quantity of RNA concentration. Looking towards tissue you had used there are high chance to get low amount of RNA concentration even you take maximum precaution and use standardize protocol. The amount you mention( 74.5ng/uL ) is enough to synthesize cDNA and further analysis of RT-PCR. I have used almost same concentration for my analysis and was able get results even at low concentration of RNA for further analysis.
Amir Mutva Hi!! What would be the main factors for low RNA concentration? I'm working with neutrophils and although we're lysing about 1x10^7 cells, the extraction is still not beeing enough for analysis... i work with proteomics and so, not at all familiar with RNA extraction methods...
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