> I am doing norhtern blots to see the RNA levels of murine=
peritoneal
>macrophage producing cytokines (IL-1a, IL-6, TNF) and T-cell producing
>cytokines (IL-2, IL-3, IFN-g). Iam using 'RIBOPROBES'. All my cDNA's=
are in p
>BSKSII+ plasmid. All cDNA's are transcribed with T7 polymerase=
except IL-3
>(T3 poly.) after linearizing with appropriate restriction enzymes.=
Iam able to
>get excellent results with IL-1a, IL-6, TNF, and IFN-g (conditions=
are; Hyb
>temp is 550 C, Hyb buffer contains 50% farmamide, Washing is high=
stringent(650
>C)- which are standerdized by me.) but I am getting lot of background=
with IL-3
>and IL-2 probes. I tried different Hyb temperatures from 42 to 600=
C If I
>increase the temperature Iam losing all signals. If I decrease the=
temperature
>Iam getting non-specific signals (High stringent washing didn't=
work). Here I
>observed an interesting correlation!. The probes for which I am=
getting
>background are 370(IL-3) and 400(IL-2) bp in lenght where as others=
range from
>800-1230 bp's. Iam also unable to strip the probe even for the=
wet membrane.=20
> Anybody had this kind of strange problem with the hybridization=
of
>short riboprobes?. What is the best way to avoid the background?.=
Should I=20
>use different kind of probes like random primed or nick translated=
DNA probes.
>( I am kind addicted to riboprobes because they so precise and accurate).=
And
>finally how to strip the membranes to reprobe the same membrane?.
> Thanks in advance.
> Sincierly
> Ravi Dugyala.
>=20
Dear Ravi Dugyala,
My mentor has given me a printout of your call for help with
riboprobes. High background on Northern blots is all too familiar=
for me.
This is a problem that I have solved using riboprobes. The only difference
is that my probe is 1244 b in length. Nevertheless, I will try to=
help you
solve the present problem.
The first thing that comes to mind is the length of your=
probes.
The high background should not be due to degradation of your riboprobes
since you get a signal with your other probes. Is it possible that=
your
IL-2 and IL-3 riboprobes are prematurely terminated due to lack of
unlabeled nucleotides? The Ambion instruction manual (MAXIscript=
in vitro
transcription kits) indicates that 2.5 micromolar of UTP is sufficient=
to
obtain a highly specific full length 0.3 kb transcript whereas 1.0
micromolar generates prematurely terminated transcripts.
What enzymes are used to linearize your IL-2 and IL-3 plasmids?
Shenborn and Mierindorf (1985) have observed a low level of transcription
from 3' overhanging ends (produced by Kpn I, Pst I, etc.). It is=
preferable
to use enzymes that generate 5' overhangs or blunt ends.
If these points are covered and that the mRNA to be measured=
is
present, your IL-2 and IL-3 riboprobes should work. Other people=
have
successfully used shorter length riboprobes. Have you tried subcloning
different regions of the cDNAs in your plasmid to use as an alternative
riboprobe?
Can you tell me how much total RNA you load/well, what kind=
of
buffer you use for gel and migration, and most importantly, what=
types of
transfers (capillary or vacuum) and membranes (Hybond N+, Schleicher=
&
Schuell, etc.) are used in your lab? I might have a protocol to help=
you
strip and reprobe your membranes.
Hope my comments are of assistance to you.
Sincerely,
Fran=E7ois Blanchette
s.immuno. at courrier.usherb.ca
