@Aletheia.
Hi Aletheia. Indeed it is highly recommended to use RNase-free water in ALL your PCR application, particularly the RT-qPCR. This is most cruical to avoid any degradations from happening to your total RNA prior to performing the reverse transcription step. You have to be aware that any contamination for your reagents (e.g. prepared from any type of purified water sources WITHOUT DEPC treatment) is a major source of variations in your obtained qPCR data, hence the importance of creating an RNase-free environment for your total RNA especially for the relative gene expression profiling applications. The way I prepare DEPC-treated water is by adding DEPC to MilliQ-quality water at a final concentration of 0.1% (v/v) and leaving stir it o/n in the fume hood, as DEPC is very unpleasant and harmful if inhaled even in small quantities. Next, the DEPC-treated water should be aliquoted into small to medium size bottles and autoclaved. I am not aware of any restrictions ofr using DEPC-treated water for the purposes I explained above. However, the only limitation I think of is that you should avoid using DEC-treated water directly without autoclaving as in this case the DEPC molecule is not degraded hence the water can be toxic especially with direct skin contact. Best wishes for your experiments. Mourad.
DEPC treatment creates a more "clean" water as it uses a chemical to destroy both DNases and RNases. Normal nuclease free water utilizes filters and autclaving to remove nucleases, but since RNases tend to be fairly hardy, it does not eliminate all nucleases. DEPC treatment would be preferred for enhance sensitivity and completely remove any potential for RNA degradation by RNases during cDNA synthesis, but nuclease free water should be sufficient for PCR. However, DEPC treatment will not affect your PCR, if you choose to use this, as long as the DEPC has been inactivated.
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