Rna quantification

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Worcesterman
Worcesterman's picture
Rna quantification

Hi,

I am currently trying to quantify extracted RNA from geobacter for use in qPCR. However, i'm not really sure what all the numbers mean from our RNA quantifier (MWG lambda scan 200). It gives A260raw, A260 Pathlength corrected, A280raw and A280 pathlength corrected for both the samples and the blanks. I know to measure purity I need a 260:280 ratio of around 2 and the A260 absorbance value for quantification. However, i'm not really sure which values to use for the equations.

Am I correct in assuming that (A260PLC-A260blank) / (A280PLC-A280blank) is what I need for purity and that the A260PLC value is what I need for quantification?

Thanks a lot

Ivan Delgado
Ivan Delgado's picture
Hi Worcesterman,

Hi Worcesterman,

Unfortunately I am not familiar with the MWG lambda scan 200 so I cannot give you a true explanation of what their different measurements mean. Still, this is what I can say with a high degree of certainty: 

1. Assuming that you are using the right instrument setting for the cuvette you are using, the pathlength corrected measurements should be the best measurements you have since the instrument has already corrected for any bias caused by your cuvette's plastic/glass.

2. To get the 260/280 ratio you need, I would divide your pathlength correct 260 measurement by the pathlength correct 280 measurement. The calculation you describe sounds fine to me too, although it seems a little more complicated than need be.

Most UV specs automatically calculate most necessary readings, such as the 260/280 ratio, but if yours does not I would try to minimize the amount of calculations you perform. Simply dividing the readings you got should do the trick. 

Best