# RNA Quantification by spectrometer - 260 nm vs 230 nm (Jun/05/2005 )

I recently did a RNA extraction from cultured cell with Qiagen RNA easy kit. During quantification with spectrometer, the report generated by our machine gave absorption at 230, 260, 280, 320nm, ratio of 260/230 and 260/280. I know the meaning of 260, 280 and 260/280 but a little confused about 230, 320 and 260/230. I thought that 230nm reads give a baseline. However, I have five samples which has ratio of 260/230 less than 1 (as less as than 0.23). Using Agilent BioAnalyser, three of them showed the normal total RNA pattern two of them have no ribosome RNA bands. Could someone tell me what 230, 320 and specially 260/230 mean (which has a range of 0.23-2.61 in my latest RNA prep)? Thanks.

-chnola-

hi that was discussed previously.
230nm is a checking of solvants contamination (phenol/ethanol/...). As the ratio 260/280 may be up tha 1.6 for good purity, the ratio of 260/230 must be up than 1.6 too. Generally it's more than 2. I would recommend to do a precipitation with ethanol absolute and NaAcetate and wash the pellet with a great volume of EtOH 70% (depends of the mass of your RNA stock ; for up to 400µg 1ml is ok).

discussions :
http://www.protocol-online.org/forums/inde...?showtopic=5968
http://www.protocol-online.org/forums/inde...?showtopic=6412

-fred_33-

320 is used as the baseline. So you can adjust your system drift. Particularly important when you have a good number of samples, some of the spectrophotometers will have significant baseline drifting. You can either re-blank or use 320 to correct.

Martin

QUOTE (chnola @ Jun 5 2005, 10:13 AM)

I recently did a RNA extraction from cultured cell with Qiagen RNA easy kit. During quantification with spectrometer, the report generated by our machine gave absorption at 230, 260, 280, 320nm, ratio of 260/230 and 260/280. I know the meaning of 260, 280 and 260/280 but a little confused about 230, 320 and 260/230. I thought that 230nm reads give a baseline. However, I have five samples which has ratio of 260/230 less than 1 (as less as than 0.23). Using Agilent BioAnalyser, three of them showed the normal total RNA pattern two of them have no ribosome RNA bands. Could someone tell me what 230, 320 and specially 260/230 mean (which has a range of 0.23-2.61 in my latest RNA prep)? Thanks.

-Pri_Martin-