Assessing RNA quantity and purity with the Nanodrop
It all starts with RNA
So, you think you have extracted some RNA - now what? Well, before you can dive into qPCR, you need to run a few quality control steps. This section may seem tedious and boring; however, the quality of your RNA will ultimately reflect upon your qPCR results. The saying 'rubbish in, rubbish out' comes to mind.
So, first things first. You need to see if you actually have RNA in your samples. You cannot presume you have RNA, you have to prove it. Not only this, but purity assessments are also vital. In other words, is your RNA sample free from other contaminants such as proteins and salts? These factors are the bad guys of downstream qPCR, since many can actually inhibit the reaction.
The video below will explain how to assess the quantity and purity of RNA in samples by using a small, yet powerful, device known as a Nanodrop.
Here are the important points to summarize this lecture:
- The Nanodrop is able to determine the concentration and purity of RNA in samples.
- The 260/280 ratio (an indication of protein contamination) should be between 1.8 and 2.0 - ideally 2.0 for pure RNA.
- The 260/230 ratio (an indication of salt contamination) should be above 2.0.