I am planning to perform microarray on some RNA samples that have low 260/230 ratio,but the 260/280 values are within the optimal range. To increase the 260/230 values closer to optimal value, I have tried to eliminate the contaminants e.g. chaotropic salts by using a column based RNA clean-up kit from ZYMO research. However, the outcomes seems to be inconsistent. Although the the 260/280 ratios stay optimal (2-2.2), but increase rate in 260/260 ratios vary considerably, and even decrease sometimes below the initial values!
All steps are being followed according to the manufacturer's instruction.
I am wondering if someone has faced similar issue and have a suggestion.
I have experienced similar problems with RNA, particularly when the concentrations of RNA are low (
i haven't used the Zymo kit, but Chaotropic salts do have considerable absorbance at 230nm, so carry over - perhaps through overloading the kit, could account for your low 260/230 readings. One good way to reduce salt carry over is old fashioned ethanol precipitation. Make yours sample to 0.3M NaoAc using Rnase free sterile 3M NaOAc at pH 4.2. Then add 2 vols of 100% ethanol (AR) mix and precipitate either at -20C for 2h, or -70C for 20 mins. Centrifuge in a microfuge at 13000g for 15minutes. Wash the pellet in 70% ethanol (AR made with RNase free water) and air dry before resuspending. If you are working at low RNA cocentrations you can visualise the pellet either by using 5ug of carrier glycogen or carrier yeast tRNA, assuming these don't interfere with your next step.
I have this kind of experience ,too.And I found when the RNA yields are very low ,the 260/230 reading is not so good,usually it's smaller than 2 whether you use precipitate method or column method.I think you should not care too much about this value sincey your 260/280 is normal.Maybe you can go further to see if your later experiments work out using this RNA sample.
@Jared the concentration in those sample is in range of 100 to 230 ng/ul, but I have few samples with higher concentrations values, showing same issue.
I am not sure if the low concentration would contribute to the issue at least in my case, but I was thinking maybe it has to do with centrifugation speed/duration, since there is not much of difference between steps described in protocols from ZYMO and QIAgene.
I have consulted with our genomics core facility staff and they mentioned this value may have not have great impact on the downstream microarray analysis, and even if it dose it can be distinguished at first QC step.
Thanks for the advises.
I also have had that kind of problem. Since you have good yields and good 260/280 you probably can just continue with your experiment. The only trick that worked for me, and not always, is to repeat the washes with 70% ethanol during the RNA clean up and then centrifuge longer to completely get ride of the ethanol.
Good luck!
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