Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Biology

Water RNA Contamination? - (Mar/05/2014 )

Hi,

I had a brain fart when I was doing my RNA extractions, the protocol said to add 95-100% ethanol to the trizol+tissue, and for some reason instead of just using the stock I decided to dilute the "100%" ethanol to 95%.  What's worse, is that I used regular diluted water from the lab and not DEPC water. Thankfully, the RNA extractions still seemed to work in terms of yield (250-500ng/ul) and RNA quality (RIN 8+), so it does not seem like the water I added resulted in RNA degradation due to RNases in the water. However, is it possible that some of the RNA that I am measuring is from the water that was mixed with the ethanol and not just from the tissue? I added ~2ul of water to the "100%" ethanol to make 50mL of 95% ethanol, and then I used 500uL of that with each tissue sample. So can that small amount of water generate some type of foreign RNA that could be a big part of the readings I am getting? Thanks.

 

-Relaxosome-

Was the water you added, DNA and RNA free water?

Standard lab water means very different things to different labs.

 

DEPC water means that it has been treated so that RNAse is inactivated.

-perneseblue-

I am not sure if it was DNA or RNA free, it was just water from the sink through one of those "white tap" filters. So I am guessing it did have RNA present, but can it be in a high enough quantity to alter the readings or interfere with gene sequencing when the RNA I am interested in is rat RNA.

-Relaxosome-

Hi,

I had a brain fart when I was doing my RNA extractions, the protocol said to add 95-100% ethanol to the trizol+tissue, and for some reason instead of just using the stock I decided to dilute the "100%" ethanol to 95%.  What's worse, is that I used regular diluted water from the lab and not DEPC water. Thankfully, the RNA extractions still seemed to work in terms of yield (250-500ng/ul) and RNA quality (RIN 8+), so it does not seem like the water I added resulted in RNA degradation due to RNases in the water. However, is it possible that some of the RNA that I am measuring is from the water that was mixed with the ethanol and not just from the tissue? I added ~2ul of water to the "100%" ethanol to make 50mL of 95% ethanol, and then I used 500uL of that with each tissue sample. So can that small amount of water generate some type of foreign RNA that could be a big part of the readings I am getting? Thanks.

 

 

 

Did you really add ~2ul of water to make 50ml ????  If this is true and not a typo, you only have 0.004% of water and your ethanol is 99.996%  so I wouldn't worry   wink.png

 

If is actually 2ml, so that the water content is 4%...  I still think the possible RNA contamination in that water will be very very small, I guess whether this is a problem or not depends on your next step.

As for the RNAses, you've obviously checked the quality of your RNA and looks good. 

-almost a doctor-

 

Hi,

I had a brain fart when I was doing my RNA extractions, the protocol said to add 95-100% ethanol to the trizol+tissue, and for some reason instead of just using the stock I decided to dilute the "100%" ethanol to 95%.  What's worse, is that I used regular diluted water from the lab and not DEPC water. Thankfully, the RNA extractions still seemed to work in terms of yield (250-500ng/ul) and RNA quality (RIN 8+), so it does not seem like the water I added resulted in RNA degradation due to RNases in the water. However, is it possible that some of the RNA that I am measuring is from the water that was mixed with the ethanol and not just from the tissue? I added ~2ul of water to the "100%" ethanol to make 50mL of 95% ethanol, and then I used 500uL of that with each tissue sample. So can that small amount of water generate some type of foreign RNA that could be a big part of the readings I am getting? Thanks.

 

 

 

Did you really add ~2ul of water to make 50ml ????  If this is true and not a typo, you only have 0.004% of water and your ethanol is 99.996%  so I wouldn't worry   wink.png

 

If is actually 2ml, so that the water content is 4%...  I still think the possible RNA contamination in that water will be very very small, I guess whether this is a problem or not depends on your next step.

As for the RNAses, you've obviously checked the quality of your RNA and looks good. 

 

Yes, sorry that was a typo, I added ~2mL not uL's. The next step is to use the RNA for gene sequencing and the tissue that was used for the RNA extraction was rat mammary tissue. So I am guessing that whatever RNA would be present in the water would be bacterial/viral in nature and not interfere with gene sequencing? Thanks.

 

-Relaxosome-