HELP HELP HELP needed with RNA extraction
/G=Rimantas/S=Plaipa/OU=GF/O=VU/PRMD=LITNET/ADMD=LITPAK/C=LT/ at mhs-relay.ac.uk
/G=Rimantas/S=Plaipa/OU=GF/O=VU/PRMD=LITNET/ADMD=LITPAK/C=LT/ at mhs-relay.ac.uk
Tue Jun 11 10:53:37 EST 1996
Jim Graham wrote:
> Rimantas has suggested that there may be actual data
> relevant to "finger RNAses". Can anyone point out any
> such references?
>
> Apparently, my own assay, which consited of tipping a
> microcentrifuge tube against my finger tip and shaking it
> -removing 10ul to a tube containing 2 ug of RNA markers
> and incubating 30 min at 37C (which was negative for me
> and a collegue) may not have been sensitive enough to
> detect the proposed low levesl of RNAse acrtivity.
>
> Any one else care to try? Rimanats suggest putting
> 20ul water on a fingertip directly and testing that for RNAse
> action on an RNA standard. (I'll do it shortly too).
>
> Jim
> J. Graham PhD
> Biology Department
> Washington University of St. Louis
>
A reply from Rimantas:
Jim probably didn't notice that my letter he references was sent
personally to him only not to Bionet for world-wide distribution. So
I present it here for other also could figure what's all about:
>
> > As Jean says, a great deal of effort which goes into avoiding RNAses is
> > probably unecessary, as they will not be found in distilled water,
> > properly cleaned glassware, new plastic, or on most people's fingers. If
> > you are spooked by the preponderance of such claims, test these
> > sources yourself with some RNA markers or rRNA!
>
> Dear Jim,
>
> as I remember, you claimed in your post a month ago that you had tested
> the fingers of your colegues and yours and hadn't found any RNases.
>
> I also tested my fingers (but not my colegues' fingers) and I did find.
> I don't like the hypothesis about difference between people on this
> aspect very much. I think the procedures and their sensitivity are
> different. I think we could compare our measurements if we relate the
> activity to the activity of certain concentration of pancreatic
> RNase. When I apply 20 microliters of buffer solution on the tip of my
> finger and wait for a minute, the activity of RNases in the solution
> aproximates the activity of 10 ng/ml of pancreatic RNase. In the first
> and presumably last so far published paper on skin RNase the authors
> used to wash whole fingers in 10 ml of solution and obtained similar
> activities (1- 10 ng/ml of pancreatic RNase). They tested 6
> individuals and all were found 'positive'.
>
> What procedure did you use ? What is its sensitivity for pancreatic
> RNase ?
The 'first and last so far' reference mentioned is:
R.W.Holley, J.Apgar, and S.H.Merrill ( 1961 #7,July)
JBC 236, PC42-PC43.Evidence for the liberation of a
nuclease from human fingers.
The second probably will be mine. I've presented a draft two weeks
ago as a poster in the first meeting of the RNA Society in Madison,
WI, USA. I hope to finish the paper and submit to press in a month (
I'm hoping so since one year). I'll be glad to send preprints for all
interested either on paper or electronicaly as it will be ready. I
add some parts of my poster at the end of this post.
I agree with Jim about distilled water, glasware, plastic but human
fingers.
I worked long time on the problem of so-called Y-A cleavages. This
thing may be not known for people which use RNA only for Notherns or
RT, but must be well known for those performing direct sequencing of
RNA, processing, investigation of secondary structure by chemical and
enzymatic probes etc. While performing such procedures frequently
small amount of products corresponding to cleavages of CA or UA
sequences appears. The extent of cleavage varies from virtually zero
to complete ruin of one's work. These cleavages are stimulated by
nonionic detergents like Triton X-100, Brij and other. Sometimes in
the presence of such compounds the cleavage may be fast enough to
degrade RNA completely in one hour. Some people claims that these
cases are manifestation of intrinsic RNA self-cleavage reaction.
There are several publications with such claims (see the end of this
post).
I spend a lot of time on these cleavages and I've arrived to the
conclusion that they are caused by RNases. Both skin RNase and
pancreatic RNase are highly specific for UA and CA sequences and
produce the same band pattern on PAGE as the alleged "self-cleavage".
(Textbooks say that pancreatic RNase is specific for pyrimidines.
That's true, but the rate of hydrolysis of YA sequences is aprox 10
fold higher than that of YG and aprox 100-fold higher that of YY (Y
means pyrimidine: U or C). Therefore after low-extent hydrolysis by
this RNase only the cleavage of YA sequences is seen).
I never had a chance to meet any of the researchers claiming that
these cleavages are produced by RNA itself neither could discuss this
issue with them. All RNA people I met didn't believe in RNA "self-
cleavage" but hadn't any substantial evidences. So the people I spoke
with in Madison at my poster were glad that at last one guy had done
that.
Because many people observes the cleavages in YA sequences, I have to
admit that skin RNase is quite widespread in human population if not
universal. What makes difference between them is their RNAs and the
work they do with RNA.
For those who would like to join this discussion and test their
fingers:
I recommend to use 100 mM of monovalent salt (e.g. ammonium or sodium
chloride) in Tris pH7.5-8 without Mg for testing RNase activities.
These conditions are optimal for RNase activity ( monovalent cations
stimulates RNases, Mg suppresses its activity by stabilizing
secondary structure).
I propose using of pancreatic RNase as a standard to relate the
observations by different people using different RNAs and different
testing conditions because it is very similar to finger's RNase (at
least to mine)
------------------------------------------------------
Abstract
THE POSSIBLE INVOLVEMENT OF RNASES IN THE SO CALLED "RNA
SELF-SPLITTING" AT Y-A BONDS
R. Plaipa and B. Juodka
Department of Biochemistry and Biophysics, Vilnius
University, Ciurlionio 21, Vilnius, Lithuania
Starting from the late 70-ies, when high resolution
PAGE was introduced into RNA chemistry, investigators
frequently observe the low level RNA self-cleavage,
mostly at UA/CA. There are several reports about the very
effective cleavage of some specific RNAs in the presence
of nonionic detergents, also in YA sequences. No kinetic
investigation neither detailed study of the causes of
this phenomena ever was published, but people tends to
believe that this cleavage is not caused by RNases,
rather that YA sequence represents some minimal ribozyme
with the intrinsic self-cleavage capability. Our
experiments, however, have shown, that RNase inhibitors
suppress this reaction completely. RNases with such a
sequence specificity are present both in humans (the skin
RNase) and microorganisms. Stable cell RNAs, e.g. mature
tRNAs are evolutionary optimized and very resistant to
these RNases in the native conditions, but e.g. pre-tRNAs
are much more sensitive. If treated carelessly, they may
appear to have the self-cleavage ability.
Although researchers started to fight the ghosts of
RNases in the very beginning of the RNA world, the rites
for killing them are based mainly on the belief yet. We
have reviewed and experimentally tested the most popular
ones (autoclaving, DEPC-ing etc.), and we have found that
its efficiency strongly depends on pH and on the
composition of the solution and may be insufficient in
practice. We have performed the screening for RNases in
the laboratory environment, and found some parts of the
surface of the workplace heavily contaminated with Y-A
specific RNase. The mode of distribution of the RNase
activity suggests, that it originates from the settling
of dead skin cells rather then from immediate touches by
bare hand, and therefore is very difficult to eliminate.
-----------------------------------------------------
In 1961 a short communication "Evidence for the
liberation of a nuclease from human fingers" by
R.W.Holley, J.Apgar, and S.H.Merrill appeared in JBC [1].
These investigators had discovered that washing two
fingers and the thumb in 10 ml of water leases
ribonuclease activity equivalent to that of 1-10 ng/ml of
RNase A. No more investigations of this RNase to our
knowledge ever were published, instead many legends,
scientific folklore, myths and unpublished observations
by many researchers were created. Various rites
(autoclaving, DEPCing etc.) supposed to scare away RNases
from laboratories were proposed and are used by various
scientists with different degree of fanaticism. Taking
care of RNases indeed is essential to work successfully
with RNA and using of special precautions helps to
prevent severe RNA degradation accidents. Nevertheless,
the shortage of information about "home" RNases and
procedures for their extermination don't allow to be sure
that RNases indeed are completely eliminated from RNA
world.
After introducing 32P labeling and high-resolution
PAGE into RNA biochemistry, a new phenomena - instability
of UA/CA sequences had been observed. Incubation of RNA
in mild conditions (pH7, 37 C)for a quite short time
(e.g. one hour) without any enzymes frequently results in
the appearance of a small amount of cleavage products,
corresponding to cleavage of mainly UA or CA sequences.
Control samples of RNA without any treatment also
frequently contains the same cleavage products. Typical
explanation for these bands, proposed in 1978 [3], is
that these sequences are fragile and hydrolyze more
easily than other. Another less popular excuse is
autoradiolysis of 32P-labeled RNAs. Most of these
observations were made on mature tRNAs. After the
discovery of ribozymes several papers appeared reporting
that some particular RNAs may self-cleave in the presence
of some organic compounds - Triton X-100 and other
nonionic detergents, PVP etc. [4,5,6,7]. Most of such
cleavages reported so far are located in UA and CA
sequences. These reactions do not require Mg2+ or other
divalent metal cations, but are stimulated by monovalent
salts and/or polyamines.
One such RNA, human pre-tRNATyr, was used in our
laboratory as a substrate for tRNA processing in vitro
when a report about its ability to self-cleave in the
presence of Triton X-100 appeared. We decided to
investigate this reaction in more detail, but this work
was hindered by very poor reproducibility of the reaction
rate. We found that the slow cleavage of mature tRNAs at
UA/CA sequences also is stimulated by Triton X100.
Unreproducibility of the rate of the cleavage, sometimes
even complete disappearance had persuaded us that this
cleavage is not an intrinsic reaction of RNA, but most
probably arises from contamination by RNases. We found
that skin RNase indeed has strong preference for CA and
UA sequences and gives nearly identical band pattern on
PAGE as the alleged "self-cleavage". Pancreatic RNase
also has a nearly identical sequence specificity.
Moreover, RNases of the similar specificity are present
in microorganisms too.
Nonionic detergents like Triton X-100 stimulates RNA
cleavage by trace amounts of RNases presumably by
stabilizing them and/or preventing adsorption by
surfaces. Anyway, these compounds do not have any
affinity for RNA and do not cause RNA cleavage when
appropriate RNases inhibitors are used.
In the native conditions, i.e. in the presence of
Mg2+, mature tRNAs are very resistant to these RNases.
Probably they are intentionally designed so since RNases
specific for YA are present in cytoplasm of both
eukaryotes and prokaryotes, though their function is
unclear yet. RNA precursors need not to be highly
resistant since they are short lived in vivo. They may
posses unpaired YA sequences highly sensitive to RNases.
Such RNAs may be unstable in the presence of minute
amounts of RNases, which do not cause any noticeable
cleavage of mature tRNAs.
We tested the most popular procedures for RNase
inactivation using pancreatic RNase (RNase A) , RNase T1
and barnase (RNase from Bacillus amyloliquefaciens), the
best studied RNases, as representatives of the RNase
world. This survey have shown that RNA world persists not
because of these procedures, but due to absence of RNases
in some very important items like distilled water, most
of chemical reagents etc. Actually, many researchers have
abandoned paranoiac implementation of all those
precautions. Nevertheless, skin RNase really does exist,
and it is the main threat for RNA in laboratory.
Microbial RNases may appear in more special situations -
because of the growth of microbes in solutions,
contamination by E.coli grown in labs etc.
Gloves helps to prevent contamination by touching by
skin RNase. The outer layer of skin, actually dead cells,
wears and in the form of invisible dust settles on all
human environment. If RNase is present in these cells,
RNase activity should be present on all things where
humans lives or work. To test this hypothesis, we checked
the surfaces of the leaned plexiglass shield used for the
work with 32P in our laboratory. These shields are rarely
if any touched by bare hand. This check was made several
times with shields used by different workers. All times
the top side, turned to face of the worker was heavily
contaminated with YA-specific RNase, while "bottom" side
essentially clean. Because none of the workers whose
shields were tested have a habit to rest the nose against
the shield while working, we must admit, that RNases do
not require direct touch for transfer from humans to
laboratory equipment. This transfer may be one cause of
so widespread RNA "self-cleavage", and the one very hard
to eliminate.
------------------------------------------------
Autoclaving
RNases unlike decent useful enzymes dont get
inactivated at elevated temperatures due to irreversible
denaturation. They as some other low molecular weight
proteins renatures very fast when temperature is lowered
below the melting point (50- 60 C for RNases A , T1 and
likes). Inactivation occurs only due to slow chemical
reactions - mainly deamidation of Asn and cleavage and
shuffling of disulfide bridges. The rate of these
reactions increases with pH. At 90 C the rate of
inactivation of RNase A is 0.13 (1/h) at pH4, 0.56 (1/h) at
pH6 and 23.4 (1/h) at pH8 [2]. Even if the rate would be 10-
fold higher at 120 C, it wouldn't be enough to ensure
complete inactivation of RNase A in 30 min at pH4.
The inactivation of RNases in common buffers during
standard autoclaving cycle (120 C, 30 min) are the
following :
Media Decrease of the activity
of RNase A, times
100 mM NH4Ac, pH4.61 4.3 ( RNase T1 - 4 , barnase - 5)
100 mM NH4Ac, pH4.9 5.5
100 mM NH4Ac, pH5.2 9.1
100 mM MES-NaOH, pH6.12 16.3
100 mM MOPS-NaOH, pH7.5 75
100 mM Tris-HCl, pH7.53 11
100 mM Tris-HCl, pH7.8 69
100 mM Tris-HCl, pH8.1 380
Tris buffer drops out of the general trend due to a
particular pH temperature dependence of Tris(-0.03 (1/K),
which means that pH of Tris buffers drops by aprox. 3 pH
units when temperature rises from 20 to 120 C).
. Although the stability of RNases A and T1 may look
impressive, they certainly don't hold the record. Since
RNases get inactivated mainly due to degradation of Asn
and Cys, their thermal stability depends on the content
of these amino acids. Some other mould RNases looks much
better in this aspect.
Sequence |Asn |Cys per |Source of RNase (PDB
code in|per |molecul |code)
Swiss-Prot |molecu |e |
|le
RNP_BOVIN 10 8 bovine pancreatic RNase
RNT1_ASPOR 9 4 T1 - Aspergillus oryzae.
RNBR_BACAM 6 0 Barnase - Bacillus
amyloliquefaciens.
RNC2_ASPCL 6 4 Aspergillus clavatus.
RNU2_USTSP 9 6 Ustilago sphaerogena
RNF1_FUSMO 10 4 Fusarium moniliforme
(Gibberella
fujikuroi).(1fus,1fut)
RNMS_ASPSA 1 4 Aspergillus saitoi
(Aspergillus phoenicis).
(1rds, 1rms)
RNSA_STRAU 2 3 Streptomyces
aureofaciens.
(1sar,2sar,1gmp,1gmq,1gmr)
RNST_SACER 2 2 Saccharopolyspora
erythraea (Streptomyces
erythraeus).
RNS3_STRAU 1 2 Streptomyces
aureofaciens.
Unfortunately, no quantitative data on the stability
of these RNases are available, but they are thoroughly
investigated in other aspects. Great body of available
structural information on many members of this RNases
family should be very helpful in engineering more stable
RNases by minimizing Asp and Cys content to zero and
creating bugs producing RNases of so far unimaginable
stability (if they don't exist yet in Nature).
--------------------------------------------------------
Alkali
RNases are inactivated reasonably fast in alkaline
media. That may be mostly useful for treatment of plastic
and metal tools used for the work with RNA. Alkaline
inactivation proceeds by the same mechanisms as the
thermal one. The inactivation is suppressed by tertiary
structure of the protein:
Conditions Activity of RNase A
remaining after 4 min,
%
100 mM NaOH, 18 C aprox 100 ( 43 % after
30 min).
100 mM NaOH, 0.1% SDS, 18 C 65
100 mM NaOH, 30 C 21
100 mM NaOH+0.1% SDS, 30 C 43
These results show, that RNase A is not completely
denatured at 18 C in 100 mM OH-, and therefore the
inactivation is stimulated by SDS, which unfolds the
polypeptide chain and exposes sensitive residues for
alkali attack. SDS is not required at 30 C, presumably
because this temperature is enough to melt RNase A
without the help of other denaturants. SDS slightly slows
down the reaction since the association of negatively
charged detergent hinders the OH- attack
-----------------------------------------------------
DEPC
Diethylpyrocarbonate inactivates RNases mainly
through modification of imidazol rings of histidine
residues. Thus, the efficiency of the inhibition depends
on the quantity and accessibility of His residues in the
protein and also on pH.
At pH4 RNase A is not inhibited completely even when
treated with very high DEPC concentrations up to 50mM
(0.1% DEPC equals 6 mM). Also, half of the activity can
be recovered by treatment with hydroxylamine, which
cleaves His modification product. This product hydrolyses
in water quite rapidly too (half-time about 25 h at 25
C pH8.0 ) so one cannot be sure that DEPC inactivates
RNases in all circumstances and inactivates them
irreversibly.
True irreversible inactivation of RNases may be
brought on only by reaction with Lys (but e.g. RNase T1
doesn't have Lys at all), Tyr and also by destruction of
His imidazol ring in the reaction analogous to the
reaction of DEPC with Ade. This later reaction is quite
slow and may be left behind by hydrolysis of DEPC.
For a long time DEPC is known to be destroyed by
Tris and so unsuitable for treatment of Tris solutions.
We have found that other buffers increase the rate of
hydrolysis of DEPC too.
solution half-time of DEPC at
20 C, min
water pH3-7 40 (170 min at +4 C)
20 mM NaAc pH5.2 23
80 mM NaAc 14
3M NaAc 1.0
100 mM NH4Ac pH7 7
200 mM Na phosphate pH6.8 10
20 mM cacodylate pH6.3 2.7
200 mM MES pH6.2 15
200 mM MOPS pH7.2 6.9
200 mM HEPES pH7.5 3.5
So the DEPC survival time is quite short in the
concentrated stock solutions of all common buffers, and
that should severely hinder its efficiency as the RNase
inhibitor.
----------------------------------------------------
The consequences of DEPCing, the smell etc.
People are frequently concerned about DEPC left in
solution after treatment with it and possibility of
inhibition of enzymatic reactions. Because of the short
lifetime of DEPC in water and water based solutions no
DEPC can survive one day or longer. In Tris buffers DEPC
in destroyed completely in few minutes. Nevertheless,
everybody who use this compound probably knows
characteristic smell which remains virtually indefinitely
after treatment with DEPC. This smell cannot be caused by
DEPC, but rather by some admixture or hydrolysis product.
DEPC reaction with water gives ethanol and carbon
dioxide:
C2H5OCOOCOC2H5+H20 ->2C2H5OH+2CO2
Ethanol also reacts with DEPC:
C2H5OCOOCOC2H5+C2H50H ->C2H5OCOOC2H5+C2H5OCOOH
giving rise to diethylcarbonate (DEC) and ethyl
ester of carbonic acid, which is unstable and decays
rapidly to ethanol and carbon dioxide.
C2H5OCOOH -> C2H5OH+CO2
This way ethanol is regenerated and overall process
is the conversion of DEPC to DEC.
Therefore water which inevitably gets into DEPC when
it is stored and used destroys DEPC catalyticaly rather
then stoichiometricaly, converting it to DEC
(diethylcarbonate). Some DEC always is present in
commercial preparations of DEPC.
We have performed analysis of several samples of
DEPC and smelling DEPC-treated water by gas-
chromatography with mass-spectrometric detection and DEC
was the only compound found besides DEPC or ethanol.
So very likely DEC causes the smell of DEPC-treated
solution. DEC is rather chemically inert and is not
destroyed by heating and autoclaving. Generally it should
not inactivate enzymes too. Therefore DEPC treated
solutions, even smelling, are generally suitable for
molecular biology. Nevertheless, there are few reports
about the adverse effects of DEPC-ing on certain subtle
process (in vitro transcription and translation [8,9])
------------------------------------------
References
[1]R.W.Holley, J.Apgar, and S.H.Merrill ( 1961 #7,July)
JBC 236, PC42-PC43.Evidence for the liberation of a
nuclease from human fingers
[2]S.E. Zale and A.M. Klibanov ( 1986 Sep 23 #19)
Biochemistry 25, 5432-5444. Why does ribonuclease
irreversibly inactivate at high temperatures?
[3]P.Carbon, C.Ehresmann, B.Ehresmann & J.P.Ebel ( 1978
#1,Oct 1) FEBS letters 94, 152-156.The sequence of
Escherichia coli ribosomal 16 S RNA determined by new
rapid gel methods
[4]R.Kierzek ( 1992 #19) NAR 20, 5073-5077.Hydrolysis of
oligoribonucleotides: influence of sequence and
length
[5]R. Kierzek ( 1992 Oct 11 #19) Nucleic-Acids-Res 20,
5079-5084. Nonenzymatic hydrolysis of
oligoribonucleotides.
[6]N. Watson, M. Gurevitz, J. Ford, and D. Apirion ( 1984
Jan 25 #3) J. Mol. Biol. 172, 301-323. Self cleavage
of a precursor RNA from bacteriophage T4
[7]H.van Tol, H.J.Gross & H.Beier ( 1989 #1) EMBO J. 8,
293-300.Non-enzymatic excision of pre-tRNA introns ?
[8]P.Gegenheimer, <pgegen at kuhub.cc.ukans.edu>post to
bionet.molbio.methds-reagnts, 24 Aug 1994.
[9]O. Brick, post to bionet.molbio.methds-reagnts, June
1995.
-----------------------------------------
Comments:
I have neither money nor time to create more stable RNases now. This
field is opened for everyone.
I couldn't found DEC in our laboratory neither get it from chemists I
know in my University. I someone has this stuff please smell it and
inform me if it smells like DEPCed solutions.
Rimantas
----------------------------------------------------------
Rimantas Plaipa
Dept. of Biochem. and Biophys., Vilnius University, Lithuania
Rimantas.Plaipa at GF.VU.LT
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