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Common causes for low RNA A260/230 ratios

RNA contamination RNeasy A260/230

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7 replies to this topic

#1 robradford

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Posted 30 July 2013 - 01:38 PM

Hi Folks,

 

I'm hoping someone might be able to answer something that has been confusing me lately.

 

Basically, I have harvested a lot of RNA from iMR90 and U2OS cells over the last two or three months using Trizol extraction, which I then clean up using a Qiagen RNeasy mini kit. 

 

My problem is, that for one particular experiment, all of the RNA I get seems to have a low A260/230 ratio on the nanodrop which I've heard is a result of phenol contamination or some other contaminant. The problem is, that when I clean up RNA from several experiments together, it's only ever this one experiment which gives low A260/230 ratios so it's not likely a problem caused during the clean up. 

 

The only thing I can think of that is different is that these samples had been isolated in Trizol, and then stored at -80oC for a few weeks longer than the rest. 

 

Would this cause this kind of absorbance value? I'm not doing microarrays with the RNA and I don't seem to have a problem with making cDNA for q-PCR so it's more of a curiosity than a burning issue I need resolved. Any input or advice would definitely be appreciated though!

 

Thanks in advance for your help/opinions,

 

Rob


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#2 memari

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Posted 30 July 2013 - 03:26 PM

Base on  my experience, for having clearer RNA, you should clean your column once more by last buffer of kits.

 

In RNeasy, you should clean column twice at the end  by RPE buffer. I suggest three times.

 

In Aurum™ Total RNA Mini Kit, you should clean column once by low salt buffer. I suggest twice.


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#3 robradford

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Posted 30 July 2013 - 03:38 PM

Base on  my experience, for having clearer RNA, you should clean your column once more by last buffer of kits.

 

In RNeasy, you should clean column twice at the end  by RPE buffer. I suggest three times.

 

In Aurum™ Total RNA Mini Kit, you should clean column once by low salt buffer. I suggest twice.

Thanks memari,

 

I'll try that out. I've worked with hundreds of RNA samples over the years and this is the 1st time I've seen this happen so I'll definitely try adding a third wash.

 

Once again, cheers,

 

Rob



#4 phage434

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Posted 30 July 2013 - 04:01 PM

A chloroform only extraction following trizol will remove much of the phenol.



#5 wdyeo

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Posted 04 August 2013 - 11:49 AM

Hi robradford,

 

I am interested to know whether you put all the isolated RNA (after extraction from Trizol) to the Qiagen RNeasy Mini, by adding the RLT buffer and the subsequent step for the clean up?

I had extracted RNA from Trizol but my ration 260/230 is too low and I am wondering what shall i do to improve the ratio?

Thank you~ :D



#6 memari

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Posted 05 August 2013 - 07:09 PM

Hi robradford,

 

I am interested to know whether you put all the isolated RNA (after extraction from Trizol) to the Qiagen RNeasy Mini, by adding the RLT buffer and the subsequent step for the clean up?

I had extracted RNA from Trizol but my ration 260/230 is too low and I am wondering what shall i do to improve the ratio?

Thank you~ biggrin.png

 

for cleaning RNA in Trizol protocol,  wash it more than once by ethanol 75%:

 

1-Add 50-100 ul of ethanol 75% to RNA pellete and move it to new vial.

2-Add 1 ml of ethanol 75% & vortex for 5-10s.

3-centrifuge 4000-7000 rcf (g) for 1-3 min.

4- repeat step 1

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For Trizol+ Qiagen RNeasy Mini, there are two ways:

1- After adding Chloroform to Trizol and centrifuging, Pass the upper phase through Qiagen RNeasy Mini and go forward.

2- Do Trizol compeletly and then add RLT buffer of RNeasy and go forward


Edited by memari, 05 August 2013 - 07:16 PM.

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Babak Memari

#7 wdyeo

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Posted 19 August 2013 - 01:36 PM

Hi Memari, 

Thanks alot for the reply ~!

Have a nice day biggrin.png



#8 memari

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Posted 19 August 2013 - 02:32 PM

For Trizol+ Qiagen RNeasy Mini, there are two ways:

 

1- After adding Chloroform to Trizol and centrifuging, Pass the upper phase through Qiagen RNeasy Mini and go forward.

 

 

After adding Chloroform to Trizol and centrifuging, Some companies suggest to add one volume of ethanol 70% to supernatant and then pass it through Qiagen RNeasy Mini clumn and go forward.


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Babak Memari





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