For MNV preparation, I inoculated MNV in Raw 267.4 cells (70 confluence) in a 6-well plate for two days, after observe CPE, I froze 6-well plate to -80C fridge for 1h and thawed at 37C water bath for 2min. This freeze-thaw cycle were conducted 3 times. then I collected supernatant in 6-well plate and centrifuged. I collected cell supernatant again to stock at -80C until use.
For MNV RNA extraction, I used QIAamp Viral RNA MINI Kit. I followed the protocol in the kit, added 140ul MNV stock to and used tips with filter. Before experiment, I spread RNase Away to clean the bench. All procedures were conducted on the bench.
After I extracted RNA, I used nanodrop to test concentration and purity. The concentration of my samples was about 50ng/ul, but the number of A260/A230 ranged from 1.16 to 0.49, much lower than 1.8. I'm not sure the most possible reason may cause contamination in my samples, or during my process.
if my MNV stock concentration is too low?
if my working environment is not clean enough?
or some hints for operation on RNA?
Hi Yuxiao Lu
Samples with such concentrations and purity are difficult to proceed with. So you have to precipitate them before reversing to cDNA.
Before taking any decision, make sure your microplate is clean by reading the absorbance of dH2O.
Low 260/230 is resulted from different causes and mostly from salts and those result form the buffers inside the kit you use.
Out contaminants also are a possible reason so you have to take care while you are isolating RNA, clean all stuff and UV them if it is applicable before starting.
All the best..
Are you sure freeze/thaw is the best idea here? One of the main problems with RNA isolation from cells is that as soon as you disrupt the cells, all sorts of RNAses get liberated from their normal cellular context and start just chewing up anything they can find. By repeated cycles of freeze thaw, you're essentially bursting the cells and letting the RNAses do their thing, multiple times.
If I were doing this, I'd removed the supernatant from the wells, wash 1x with PBS, and then literally just dump trizol (or equivalent) straight into the wells and squooge around with a cell scraper. This would be what I use for RNA isolation. Addition of trizol (which contains chaotropic salts and bucketloads of phenol) unfolds everything near enough instantly, and stops RNAses working.
The other issue you have is that 260/230 is a ratio, so a low value can reflect salt contamination (high 230), low [RNA] (low 260) or some combination of the two. And you don't have a lot of RNA (unless you have a large volume at 50ng.ul-1).
You can reprecipitate your RNA, as Mohamed Khashan suggests. Reprecipitation usually brings down 'most' of your RNA but much less of your contaminating salts, and also allows you to decide what an appropriate volume would be for dissolving the pellet (i.e. a tiny pellet could be dissolved in 10ul, while a massive pellet might warrant 50ul).
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