I am isolating RNA from whole blood for micro-array purpose. For this, I have to perform DNase treatment for purification of collected RNA. There are some commercially available reagent named Absolute RNA wash solution for this purpose. But, My question is, how can I prepare this solution at lab?
What method do you use for your RNA extraction? if you use commercial kit, the RNA Wash Solution are always included. In my experience, poor RNA quality mainly caused by tissue collection and homogenization. With RNA Wash Solution just fee safe to used those are in the kits. If you use traditional trizol method, use 75% alcohol to wash also fine. You need to adjust how many time washing based on the ration of A260/230 of your RNA from Nano drop measurement (I suggest to use Qbit in the case using RNA for sequencing), I suggest to wash 3 to 4 times. Hope it helps.
Dear Amber,
Thanks for your reply. I use a commercial kit for RNA isolation. Actually, I asked, how can I prepare the wash solution at lab instead of commercial?
for microarray you need highly purified RNA So, hand made RNA wash solution may be risky and RNA is easily degraded
Absolute RNA is a component of an Applied Biosystems RNA isolation kit. If you have the kit then it comes with the solution, but it's useless without the kit. It will not work with a kit from a different manufacturer. It is designed to work only within the context of the kit chemistry so it has no value on it's own.
A protocol for removal of gDNA from RNA preparations prepared using trizol is as follows:
-First prepare RNA using trizol reagent according to the manufacturers directions or use RNA prepared using another kit/method.
-Prepare the following 10 X buffer using RNASE-FREE MATERIALS!: 100 mM Tris-HCl pH 7.5, 25 mM MgCl2, 1 mM CaCl2, 10 mM DTT, or use the DNase I buffer that is supplied with the DNase I.
-Mix the following in an RNase-free microfuge tube:
up to 100 ug RNA
50 uL 10 X buffer
50 U Rnase Inhibitor
Water to 495 uL
5 U DNase I
Mix and incubate at 37 C for 30 min
Add 10 uL of 0.5 M EDTA pH 8.0
Extract with 500 uL of 5:1 phenol:chloroform pH 4.3 (also known as acid phenol)
Centrifuge 15,000 g 5 min 4 C
Remove supernatant and extract with 500 uL of 24:1 chloroform:isoamyl alcohol
Centrifuge 15,000 g 5 min 4 C
Remove supernatant and add 55 uL of 3 M sodium acetate pH 5.2
Add 555 uL isopropanol
Place on ice 15 min
Centrifuge 15,000 g 15 min 4 C
Remove supernatant and wash the pellet twice in 1 mL of ice-cold 75% ethanol (centrifuge 5 min 15000 g 4 C in between washes)
Allow pellet to dry just to the point where no droplets of liquid are visible. Allowing the pellet to dry completely will make the RNA impossible to resuspend.
Resuspend in RNase-free water.
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