Dear Kadriye Guven ,

It is recommended to measure the concentration of total RNA after aliquoting, as the process of thawing and aliquoting the RNA can affect its quality and concentration. Measuring the concentration of each aliquot after thawing can help ensure that you are using the correct amount of RNA for your qRT-PCR, which can help reduce variability in the CT values. If you rely on the first concentration taken before aliquoting, there is a chance that the aliquoting process may have affected the quality or concentration of the RNA, which could result in lower accuracy in your qRT-PCR results.

Nicolò M. Villa Thanks for your comment. That makes sense.

That's the comment from Qiagen: In theory, the RNA concentration should not change after storage, and it should be the same for all aliquotes and (very) similar to the one measured first. It is usually unnecessary to remeasure each time, and sometimes the volume is so small that it can be done only once. Purified RNA can be stored at -20°C or -80°C, which should be fine. If RNA concentration is very different on the aliquote vs the first measurement, then it can be that the RNA is degraded."

I found that aliquoting the RNA affected my samples' concentration and quality. Some samples' concentration was very similar to the one measured first. Remaining samples which were low concentration after the isolation was affected.

Best wishes,

it is better to use fresh RNA always and plan your experiment like this, however, every time measuring concentration after thawed it will help to achieve the

accuracy.