I am little bit confuse between the composition and function of Nuclease-Free Water and Rnase-Inhibitor in RNA extraction .Does Nuclease-Free Water act as Rnase inhibitor ?
Hello Mena Motaz
Nuclease-free water is free of endonucleases, exonucleases, and RNases. This is a type of water which is deionized, filtered, and autoclaved, and fit to be used for RNA extraction.
On the other hand, RNase Inhibitors work by binding strongly to RNases, preventing them from degrading vulnerable RNA molecules during experimental work such as RNA extraction.
During RNA extraction, the ribonuclease enzymes present in cells and tissues are released which can rapidly degrade RNA. RNases act without cofactors and are stable enzymes. Therefore, the most important step during RNA extraction would be to inactivate endogenous RNase. So, you may find in many protocols of RNA extraction the usage of strong denaturants such as guanidium salt which disrupts the cells, solubilizes its components, and denature the endogenous RNase at the same time.
So, during RNA extraction, it is advisable to use both, nuclease free water to prevent the introduction of RNases during RNA extraction as well as introduce RNase inhibitors to inactivate endogenous RNase.
I need to clarify that nuclease free water does not act as RNase inhibitor. Both of these serve two different purposes.
Best.
I get it now. I am very grateful to you. Your answer is so much clear to me. Thanks.
Malcolm Nobre
Hello,
Nuclease free water (NFW) is a water that is marketed after making it free of contaminants like DNases, RNases, endonucleases, etc. Hence, this is used in RNA purification to prevent contamination and degradation of the RNA to be isolated by any nuclease. NFW is used instead of normal water to assure that "labile" RNA is not degraded during or after isolation. This needs to stored carefully.
RNase inhibitors prevents the function of RNases present in the cell or outside the cell. These can be used during RNA isolation to prevent the action of RNases present in cell.
Both have similar roles, i.e., to prevent degradation of labile RNA but they serve different functions. NFW prevents contamination during RNA isolation since it has no RNase, DNase, etc. RNase inhibitors assures inactivation of endogenous or even omnipresent RNases.
Hope this helps.
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