Table of Contents Table of Contents
Previous Page  46 / 68 Next Page
Information
Show Menu
Previous Page 46 / 68 Next Page
Page Background

page

46

Lab Times

1-2015

Methods

Lab Hint

S

ome laboratory reagents are so fa-

miliar that we take them for granted

and yet these molecular workhorses

can occasionally surprise us, yielding unex-

pected and potentially misleading results in

standard procedures.

We were not expecting RNase A, an ex-

ceptionally well characterised enzyme, to

do anything other than degrade the RNA

in our samples but we actually observed

RNase A removing substantial quantities

of DNA under common experimental con-

ditions. This activity confounds the results

of basic RNase susceptibility assays and has

the potential to introduce sequence bias

during DNA purification that would intro-

duce artefacts in genome-wide analyses.

While searching for transcripts from

mouse major satellite repeat sequences by

Northern blot, we observed strong signals

in RNA samples derived from cultured fi-

broblasts. To ensure that these signals

did not represent contaminating genom-

ic DNA, we performed a routine digestion

with RNase A and the signals disappeared

as expected. Other results, however, were

puzzling as the major satellite signals were

completely removed by DNase I but not by

some other ribonucleases.

Puzzling results

This unexpected result led us to sus-

pect that the signals stemmed from genom-

ic DNA and that RNase Amay possess an in-

nate activity against DNA. Confirming this

suspicion, we found that RNase A readi-

ly removed the signal of a major satellite

PCR product spiked into the assay (see Fig-

ure). This ability was not unique to a par-

ticular batch of RNase A and multiple oth-

er brands, including certified DNase-free

RNase A, displayed the same activity.

Anecdotal reports do suggest that RNase

A can degrade DNA but such reports receive

little credence as the well-understood cata-

lytic mechanism of RNase A absolutely pre-

cludes DNA cleavage. It was shown long

ago, however, that RNase A can bind DNA

and our experiments reveal that RNase A

holds onto bound DNA with such ferocious

tenacity that DNA-RNase A complexes are

not dissolved by

phenol:chloroform

. When

phenol:chloroform

extraction is used to

purify DNA samples treated with RNase A,

these complexes partition efficiently to the

organic phase and the DNA is lost.

DNA binding while ‘breathing’

Loss of DNA is not a peculiarity of ma-

jor satellite sequences as a DNA molecu-

lar weight marker was also efficiently re-

moved by RNase A. However, we predict

that there will be a sequence bias as RNase

A binds to single stranded regions of DNA as

it ‘breathes’ (a process in which regions of

the duplex transiently open), which occurs

at a sequence-dependent rate. The quan-

tities of DNA removed are also not small;

1µg RNase A completely removes at least

50ng DNA from a sample, so a few µl’s of

commercially available RNase A (usually

10-20mg/ml) added to a DNA sample could

remove micrograms of DNA.

What to do?

There is no easy solution to this problem

as proteinase K digestion was insufficient to

completely release the bound DNA. The ef-

fect can be partially suppressed by increas-

ing the salt concentration but DNA remov-

al still occurs. Column-based purification

methods should avoid DNA loss but may

suffer the opposite problem of co-purifying

the RNase A leading to DNA migration de-

fects, which we also observed.

We recommend that RNase A treatment

is avoided entirely for critical DNA purifica-

tion applications, particularly on low abun-

dance samples. RNase T1 did not show the

same effect in our hands and should, there-

fore, be usable for RNA removal post-puri-

fication.

Of course, RNase A finds many applica-

tions outside DNA purification; we suggest

that care is taken when interpreting any as-

say involving RNase A, to ensure that the re-

sults cannot be explained by strong binding

of RNase A to DNA or chromatin.

Federico Donà and Jon Houseley

(

The Babraham Institute, UK)

Tips and tricks of the trade

Expect the Unexpected

Researchers thought they knew most everything about RNase A: the RNA degrading enzyme was purified in the

1940s already, the structure was solved in the 1960s and the catalytic reaction mechanism can be found in detail

in every biochemistry text book. But RNase A may still catch you by surprise.

Do you have any useful tips?

Contact us at:

editors@lab-times.org

Get your own copy

– it’s free!

Lab Times

is free of charge for non-profit institutions all over Europe. The life science journal is distributed to scientists and lab staff for free* wherever they work:

at universities, research units, private and public research institutes, etc. You are welcome to order multiple free copies for your department (just let us know the

quantity). Any enquiries to Lj-Verlag,

Lab Times

, Merzhauser Strasse 177, 79100 Freiburg, Germany, +49-761/35738 (fax), or

subscription@labtimes.org

(email).

For online subscription see

www.labtimes.org

or

www.labtimes.org/labtimes/subscribe.

An e-paper version is also available at our website.

* For companies and personal subscriptions (if you want us to send

Lab Times

to your home address) the subscription fee is € 27.- per year (7 issues).

A major satel-

lite PCR product

is efficiently re-

moved from an

RNA sample by

RNase A treat-

ment and phe-

nol/chloroform

purification.

RNase A from

three different

manufacturers

was tested.