260/230 and RNA-seq library prep

[TAR76]

Hi,
Does anyone have experience of low 260/230 values for RNA impacting adversely on Illumina truseq total RNA library prep for RNA-seq? I have good 260/280 values and adequate quantities of RNA but low 260/230s. I have heard various opinions about the utility of 260/230 ratios but wondered what other experiences were? Thanks!

[pmiguel]

Hi,
Does anyone have experience of low 260/230 values for RNA impacting adversely on Illumina truseq total RNA library prep for RNA-seq? I have good 260/280 values and adequate quantities of RNA but low 260/230s. I have heard various opinions about the utility of 260/230 ratios but wondered what other experiences were? Thanks!

My opinion is that since you are probably using a Nanodrop that does all the work of producing an entire spectrum for you, why not just look at that rather than use a ratio metric designed back when you had to manually change wavelengths on most spectrophotometers?

If you did, you might have a chance of identifying the contaminating substance by its UV absorbance spectrum.

If you are willing to consider the spectrum, check out my thread on this subject:

http://seqanswers.com/forums/showthread.php?t=21280

The short answer is that the most common culprits for causing your A230 to skew higher are:

(1) Acetic acid and salts thereof.
(2) Guanidine -- HCl and Isothiocyanate salts have different spectra
(3) Betamercaptoethanol.

Note that I don't include phenol! Phenol has a higher absorbance at A260 than at A230, so it can't be responsible for decreasing your 260/230 ratio. Oh, unless you are using Tris-acetate buffer to equilibrate your phenol. But then it is the acetate, not the phenol decreasing your 260/230 ratio.

--
Phillip

[LTJensen]

Yes, we have seen that low 260/230 ratios negatively affects the TruSeq total RNA prep. When we experience low ratios, we always do a wash on a MinElute filter to improve purity.

[pmiguel]

Yes, we have seen that low 260/230 ratios negatively affects the TruSeq total RNA prep. When we experience low ratios, we always do a wash on a MinElute filter to improve purity.

Ah, a ritual of purification. I was in a lab once that had a shrine to the "oligod". Lab members would burn pieces of nitrocellulose there before an important molecular experiment.

Here is something to think about -- what about all the "contaminants" that don't absorb UV? How are you detecting those?

--
Phillip

[LTJensen]

I see your point pmiguel, nevertheless the ritual worked for us... When ratios are fine, we never have problems with that particular protocol.