RNA quantification

DNA qPCR from DNA Template
1

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

2

You are correct in using the pathlength-corrected values and subtracting the blank readings for both purity and quantification. The A260PLC is used for quantification, and the ratio of (A260PLC - A260_blank) / (A280PLC - A280_blank) is used to assess RNA purity.