RNA sample extracted from the leaf of a medicinal plant with a manual method has the following characteristics, and dissolved in nuclease free water.
How can I improve the quality of this sample? Is there a way or should I do another extraction from the beginning? Can quality of this sample be increased by using RNA column without reducing its concentration? Or is it possible to increase its quality due to the fact that the extraction process has ended?
RNA concentration: 1500µg/ml
A260/280: 1.80
A260/230: 1.68
Volume: 22 ul
Buffer: Nuclease free water
As RNA goes, that's really not that bad. My benchmark for 'cDNA synthesis grade' RNA is a 260/280 of 1.8+ and a 260/230 of 1.7+, so if I had a sample like yours I'd probably keep it as is.
You've also got a lot of it, so I'm not really sure what the problem is.
If you want to improve the 260/230, you can reprecipitate it:
Make up the vol to 500ul with H2O
add 50ul of 3M sodium acetate, pH5.5
10ug of glycogen (optional, increases recovery)
500ul of isopropanol
Room temp for 20mins, spin at full speed in a chilled benchtop microfuge. Wash 2x with ice cold 75% ethanol, all pellet to dry, resuspend in water.
You'll lose some RNA in the process, but you'll lose more salt, so the 260/230 ratio should be 2+ afterwards.
Again, though: 1.68 isn't that bad.
Sir, I believe that even I must have had the same problem. I had a low cell count (around 10^5 cell/mL) and I had added about 0.5mL of Trizol. I did have a poor yield whose range was somewhere around 1.36-1.56. So did the 260/230 ratio turn out to be extremely low (around 0.15 to 0.49). at the risk of being unruly cautious, I fear that 3M Sodium acetate might render my RNA highly salty (although I do understand its purpose). However, I did look into the suggestions of Thermo Fisher where they suggested to add 0.2M Na+ ions or 0.5M NH4+ ions. Will that suffice if I choose to use 0.2M NaAc or it will be alright to use 3M of the same for the low cell count ?
Also, should sodium acetate that is made, should it be made using DEPC-treated water and autoclaved just to be on the safer side ? I would like to try this protocol ! Could you please help me out ?
Make it RNAse free, yes. I usually autoclave mine.
And remember, you're only adding 50ul (to a 500ul vol), so the final concentration is 0.3M.
The Na+ ions help stabilise the phosphate backbone, helping the RNA to precipitate. Salt is lost in subsequent wash steps (2x washes with 75% ice-cold ethanol) and I've never had any problems with downstream applications due to sodium.
Sir, I would really like to thank you for your suggestions ! It worked !! I had samples with abysmally low 260/230 ratios of 0.27 and 0.30 which escalated all the way to 1.37 and 1.06 respectively! I agree that these values are still not the preferred ones, but I really feel happier to have desalted out the salty RNA to a certain extant ! Thank you so much sir !!
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