Red Pill or Blue?

Carrying out science often involves many difficult decisions! I see it all the time in RNA protocols – the “gracious” option of using purified water or Tris-EDTA (TE) buffer to dissolve (or elute, if you are using column purification) RNA. When I was trained in assessing RNA using UV spectrophotometry, graduate students just shrugged at me when I asked which solvent was best to use. Given the ease of using it, I gravitated toward good ol’ H2O. For quantifying and checking the purity of RNA by “spec”, however, I later found out that TE is definitely the best option. By showing you some of my data, I can illustrate why!

Same Samples, Different Numbers When Assessing RNA!

Many people say you should use TE to check your RNA to ensure stability in the results. In fact, I started looking more into water vs. TE once my lab mate saw purity numbers that fluctuated depending on how much she diluted her RNA in water (weird right?). Stability, as I later found, is only one of the benefits of using TE. Firstly, check out the concentration and absorbance ratios I got using identical spleen-extracted RNA samples diluted in either diethylpyrocarbonate (DEPC)-treated water or 1x TE buffer (Table 1).

Sample #RNA (Water)
Conc. (μg/μL)
RNA (Water)
Conc. (μg/μL)
12.08 1.751.692.1

Table 1. Comparison of Different Diluents on RNA Assessment. RNA samples are diluted 1:20 in water (Water) or 1:20 in TE (TE).

In spectrophotometric analysis of RNA, the “rule of thumb” is that pure RNA has an A260/280 ratio of 2.1. If you are doing qPCR or RT-qPCR, as I am, you might not even consider using RNA with an A260/280 ratio below 1.8. Sample #1 in the results above might raise an eyebrow, depending on how you round off your values. Surprisingly, the same sample, gives a “perfect” ratio of 2.1 when analyzed in TE buffer. The other main difference to note is the consistently higher RNA concentrations when analyzed in water. If I were to use these values, would I be overestimating the amount of RNA I have? How can the diluent make such a difference?

pH Matters!

The fact that DEPC-treated water was used is important because this water in particular tends to have a low pH (~5.0), whereas TE buffer has a much more alkaline pH (7.0-8.0). After a a little digging, I found out that pH can influence the absorbances measured at 260 nm and 280 nm, affecting quantification and purity assessments, respectively.1,2 If you aren’t using DEPC-treated water, you might not have anything to worry about. Unfortunately, measuring the pH of very pure water is tricky business. If you don’t seem to be getting weird numbers like me, there is probably no need to panic! If you want to double-check whether pH is affecting your measurements, you can either compare readings in your water to those you get using pH 8.0 1xTE or see what happens to your readings when you serially dilute your RNA in your water.

In my own data, multiple parameters of the RNA analyses were influenced by a simple diluent change, most notably the purity assessment. Other sources also report that the A260/280 ratio is most sensitive to changes in pH, going from 1.8 at a pH of 5.4-5.5 to > 2.0 at a pH of 8.0.2 If you carry out your spec measurements in low pH water, you might conclude that your RNA just isn’t up to scratch! It is therefore recommended that analysis be carried out in a buffer near pH 8.0 for the most reliable and accurate absorbance values.2

Also, you might want to consider the protective effects a higher pH gives to RNA during subsequent reverse transcription and/or PCR steps. Hydrolysis of RNA occurs more rapidly at higher temperatures – an optimal pH can help prevent this. Finally, the optimum pH range for reverse transcriptase itself is usually between 8.0-8.5. Therefore, TE would be the best choice to prevent lowering the pH in your reaction.

TE: The Safe Bet!

The good news is that if you’ve been using low-pH water, your RNA might be much purer than you thought! If anything, you’ll get more consistent values that don’t depend on your dilution factor, like we saw when we added more water. Just make sure that whichever diluent you use, you also use that same diluent as a blank. Happy spec’ing!

Additional Reading

  1. Wilfinger, William W., Mackey, Karol and Chomczynski, Piotr. (1997) Effect of pH and Ionic Strength on the Spectrophotometric Assessment of Nucleic Acid Purity. Biotechniques 22:474-481.
  2. Aranda IV, Roman, Dineen, Shauna, M., Craig, Rhonda L., Guerrieri, Richard, A., Robertson, James M. (2009) Comparison and evaluation of RNA quantification methods using viral, prokaryotic, and eukaryotic RNA over a 104 concentration range. Analytical Biochemistry 387: 122-127.