real-time PCR summary

Nobody nobody at hgmp.mrc.ac.uk
Mon Sep 24 11:55:18 EST 2001


<x-flowed>My recent request for information about using Real Time PCR in
Arabidopsis generated more requests to repost the replies than actual
replies, so here's what I've received:
original ?:
We have begun experimenting in our lab with Real Time PCR as a
>=20 quantitative expression analysis tool.  Although we work with
>=20 Medicago truncatula, we rely heavily on the Arabidopsis community
>=20 when developing new techniques.  However, I've been unable to find
>=20 published reports of the use of Real Time PCR in Arabidopsis.  I may
>=20 be using the wrong search terms, but I'd appreciate any information
>=20 about published reports that use this technique.
>
>=20 In addition, I'd be interested in hearing from anyone who's presently
>=20 using this technique in Arabidopsis, published or not.  What gene do
>=20 you typically use as a control?  What primers are you using in
>=20 Cybergreen assays?  I've seen reports in other organisms of using
>=20 actin as a control, but I've heard that actin levels can be
>=20 influenced by plant hormones, and since we're specifically looking at
>=20 things affected by plant hormones, actin wouldn't be the appropriate
>=20 control.
>
>=20 Any suggestions, comments, or caveats are appreciated.  If there's a
>=20 general interest, I'll share the responses with the newsgroup.
>=20 Thanks,

responses:
It's been a while since I developed TaqMan assays primarily for plants, so
perhaps some others will give you a more up-to-date response.  I used the
nuclear component of RuBisCO for normalizing gene copy number when testing
for number of marker insertions in transgenics.  Particularly in maize there
is only one gene for the small subunit, with two mRNAs.  I made primers to
the 3' portion so I would amplify only an apparent single-copy gene (works
for either mRNA or genomic).  I developed a two-color assay to
simultaneously detect both the control gene and the transgene so I could
determine copy number.  Unfortunately I'm not sure about single copy genes
in your plant, but perhaps you know of one.

David A. Knorr, Ph.D.
Senior Scientist
LYNX Therapeutics
dknorr at lynxgen.com
****************************************************************************=
**
We bought the BioRad icycler last fall- it's been working pretty well
for us. We're using UBQ10 primers as our control primers.. I haven't
seen any published reports either.

We've had two major problems so far- general contamination (we see small
amounts of amplification in our negative control) and genomic DNA
contamination of our cDNA samples.  Our primers span an intron so we run
the products out on a gel and often see two different bands. So, we've
established a clean area for setting up our Real time reactions- and
contamination has gone down. And, we now routinely treat our RNA samples
with DNase.
Erin L. Connolly
Assistant Professor
Department of Biological Sciences
University of South Carolina
Columbia, SC 29208
****************************************************************************=
**
Julia,

I have been using TaqMan real time PCR in Arabidopsis using ACT2 as an
internal control; this suits us fine since this actin isoform is highly and
uniformly expressed in aerial green tissue. I have developed an ACT2
primer/probe set that works specifically (does not pick up other ACT
isoforms) and is cDNA specific. The set was designed to work on Col-0, Ler
and Ws-0 ecotypes of Arabidopsis.

Details will be published shortly (1 Oct 2001) in a paper coming out in EMBO
Journal entitled "Direct Interaction Between the Arabidopsis Disease
Resistance Signaling Proteins, EDS1 and PAD4".

Hope this helps,

Dr.Bart Feys
Sainsbury Laboratory
John Innes Centre
Colney Lane
Norwich NR4 7UH
UK

Email   bart.feys at bbsrc.ac.uk

**********************************************************************
I am responding to your email to the arabidopsis community.
An article I have found helpful is:

"Quantitative Reat-Time PCR Assay for Determining Transgene Copy
Number in Transformed Plants"

David J. Ingham, Sandra Beer, Stephenie Money, and Genevieve Hansen

BioTechniques 31:132-140 July 2001

As a control for my Cybergreen assays I have been using primers to Ubiquitin=
=2E

Good luck,
Brenda
Brenda.B.Parson at Dartmouth.EDU
************************************************************************
we have published a paper using real time pcr for arabidopsis.  we used
rRNA for the control in RT PCR, but tubulin and actin in Northerns.

Levin, J.Z., A.J. de Framond, A. Tuttle, M.W. Bauer, and P.B. Heifetz (2000)
Methods of double-stranded RNA-mediated gene inactivation in Arabidopsis and
their use to define an essential gene in methionine biosynthesis  Plant Mol
Biol., 44: 759-775.

Joshua Z. Levin, Ph.D.
Syngenta
3054 Cornwallis Road
Research Triangle Park, NC  27709-2257
Email:  joshua.levin at syngenta.com
************************************************************************
I'm a grad student at Iowa State and I've been doing some real time
PCR in maize.  Specifically, looking for genes expressed during
kernel development.  It's worked pretty well so far. I don't know of
any published reports.  I had difficulty finding any when I was doing
a search several months ago.  If you come across any from other
people, I'd be interested to see them! I've been using a GeneAmp 5700
doing the SyBr green assay (vs. Taqman).  I haven't had any problems
with non-specific amplification, so with my particular gene, this was
a good choice.  I would recommend using the 18s rRNA for your
controls.  A rep I talked to at Applied Biosystems had did a study
looking at numerous control genes in mammals including 18s, G6PDH,
actin, etc. and it turns out that 18s is really the best at remaining
a consistent indicator of RNA levels across many samples.  I designed
my own primers for the maize 18s, but later found out that the 18s
control primers sold by Applied Biosystems (as a kit) also work on
maize.   In their product literature, they only guaranteed animal
systems, I think.  I would therefore assume that they would also work
on Arabidopsis.

There's lots of stuff I had to learn along the way, but once I
figured out all of theory, it was all very easy.  Let me know if you
have any other questions.
Jason R. Dinges
Dept. of Biochemistry, Biophysics, and Molecular Biology
Iowa State University
2182 Molecular Biology Building
Ames, IA 50011
Ph: 515-294-8202
=46ax: 515-294-0453
****************************************************************************=
***
I saw your messages concerning realtime PCR in the Newsgroup. There aren't
actually so many publication using plant material in realtime PCR
experiments. We use in our lab the LightCycler which differs somewhat from
the Taqman technology from Applied Biosystems.

Publications:
There are no publications using Arabidopsis to my knowledge...

else:

>=20 Cooper B (2001) The Plant  Journal, 26(3), 339-349
This Publication used Chenopodium amaranticolor as a system

>Roberts CA et al. (2000), J Virol Methods  Jul;88(1):1-8

>Weller SA et al. (2000) Appl Environ Microbiol  Jul;66(7):2853-2858

>Vaitilingom M et al. (1999) J Agric Food Chem  Dec;47(12):5261-5266

About controls:

In my experiments I use indeed the actin2 gene as a control. I perform
stress treatments and also some phytohormone treatments on Arabidopsis. The
absolute changes I observe are not strong (1.3 to 3.2 fold), but the changes
might well come from the different RT's  itself. The induction of the genes
compared to the induction of actin is dramatically: Stress specific
induction of stress induced gene can go up to 300 times!! So in my hands,
test if you have strong changes in actin2 absolute expression, if not, go
ahead (You also can test this by Northern blotting to exclude the RT step).

A good paper for quantification is in my view also this one:

Pfaffl MW (2001) Nucleic Acid Res. (29)2002-2007
Oliver K=FCrsteiner / Ph.D.student
Institute of Plant Sciences
University of Berne
Altenbergrain 21
CH-3013 Berne, Switzerland
oliver.kuersteiner at ips.unibe.ch

Hope this helps!
**********************************************************************
**********
Julia Frugoli
Asst. Professor
Department of Genetics & Biochemistry
Clemson University
122 Long Hall
Clemson, SC 29634

PHONE (864) 656-1859
=46AX (864) 656-6879
webpage: http://www.clemson.edu/~jfrugol
**********************************************************************
**********

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