Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

RNA concentration suddenly drops - (Jun/18/2013 )

Hi Everybody,

I had an odd occurance the other day. I was collecting RNA using the RNEasy Qiagen kit...and I speced it as soon as I was done for RNA, and I got about 400 ng/ul...the next day I come into lab, get ready to do some qPCR and I spec it again, and the concentration suddnely dips to 30 ng/ul! I made sure the spec was working fine with some other samples of known concentration...I made sure the lamp was working, and I also made sure I was pipetting properly...

What could it be?!


PS: It only dramatically decreased with 4 out of 8 samples too, so it wasn't across the board!


How did you stored it?
In my experience RNA degrades easily but I am not sure if from one day to another can degrade such this (depending on the storage method)


That is a pretty significant decrease. Are your RNA concentrations more like the first day or the second day?


Today I did some RNA extraction and I found that if you mix Trizol or RNAzol with column base kits you have a cleaner RNA with less tendency for decreasing RNA.
RNAzol + Aurum Total RNA Mini Kit gave me the cleanest RNA. I mean Aurum Total RNA Mini Kit gave me clean(260/280= 2.08, 260/230=2.11) RNA but after 30 minutes I had a lot of decrease of RNA. But in RNAzol + Aurum Total RNA Mini Kit, Total RNA decreased from 98 to 96 microgram, but in Aurum Total RNA Mini Kit alone I saw a decrease from 102 to 80.

I suggest you to do a RNA extraction first by RNAzol (it is much more cheaper than Trizol )
, then after twice washing its RNA pelete (I suggest moving RNA pellete to new vial between these two washing steps) by 70-75 % ethanol, start using a column base kits to clean it again.

I have used RNeasy (Plus). It is completely similar to Aurum Total RNA Mini Kit but more expensive.
They start with adding 350 ul lysis buffer contanning 1 % Mercaotpethanol. Then adding 350 ul 70-75 % ethanol and passing it through a column and then washing and eluting.