comparisons with NGS dat? Re: [Arabidopsis] Re: good set of "housekeeping" genes in AT?

Ann Loraine via arab-gen%40net.bio.net (by aloraine from gmail.com)
Mon Oct 5 07:42:18 EST 2009


Thank you for this excellent advice!

I've been following this thread with great interest in hopes of finding good
controls for expression experiments.

Has anyone assessed the behavior of "invariant" (in quotes because they may
still vary under some treatments) genes using next-gen sequencing data?

There are a several new Illumina data sets available in the short-read
archive (from Oregon State) and my lab has a data set from a 454 sequencing
experiment (longer reads) comparing a treatment and control.

We were thinking of using the "invariant" genes to calculate a normalization
factor across the data sets so that we could more confidently estimate
differential expression, and then, of course, follow up with another gene
measurement method (e.g., qRT-PCR) to confirm the results.

Knowing which genes really do not vary (much) under our conditions of
interest will be critical.

Best,

Ann Loraine

On Sat, Oct 3, 2009 at 7:30 PM, Eve Wurtele <evewurtele from gmail.com> wrote:

> If you are concerned with more than developmental expression, 100 genes
> that
> vary least across about 1000 Affy chips (500 samples) from AtGenExpress are
> described in Mentzen 2008. (The chips include the developmental series,
> plus
> a variety of environmental and genetic perturbations).  The reference as
> well as the actual "steady" gene list are below.  BMC Plant Biol. 2008 Sep
> 30;8:99.  Regulon organization of
> Arabidopsis.<
> http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=18826618
> >
> *Mentzen WI*<
> http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=18826618
> >,
> <http://goog_1254598750316>*Wurtele
> ES*<
> http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=18826618
> >.
> Listed at supplementary "*Additional file 5.* Genes with the most steady
> expression"<
> http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=18826618
> >
> Two quite steady transcripts with a high overall level of accumulation are
> :
> AT3G56460 and AT4G34110 (These transcripts have about 5-fold and 7-fold,
> respectively, higher accumulation than the mean value of the genes on the
> chips, which is set to 100)
> The genes with most steady expression comprise a fairly uniform mixture of
> metabolism, regulation, transport, and "unknown" functions.
>
> However,  no "steady" "housekeeping" genes on the affy chip are completely
> steady.  Thus, it makes sense to be sure the particular conditions you are
> testing (say "heat stress") are not the ones under which the "housekeeping
> gene" varies. For each gene, there are a couple conditions/in a couple
> chips
> out of the 500 in which there is variation (say 2-fold greater or less than
> the mean). You can use
> MetaOmGraph<http://metnetdb.org/MetNet_MetaOmGraph.htm>to visualize
> the conditions where there is a bit more variation.  Download
> (free- funded by NSF-Arabidopsis 2010), a window will open. download
> Affy.ath1.data1 project, clicking the blue +to create a new gene list, and
> paste the locus IDs shown below into a the gene list after clicking import.
>
> (that's what I did to get the particular three genes listed above, its
> quick
> once you've learned the software)
>
> *Locus ID- Most steady expression- Mentzen and Wurtele, BMC Plant Biology
> 2008*
> AT5G12370
> AT4G15415
> AT4G15475
> AT4G17430
> AT1G73440
> AT5G11040
> AT5G28850; AT5G28900
> AT5G21040
> AT4G37130
> AT3G56830
> AT1G16570
> AT5G16280
> AT5G24830
> AT5G65540
> AT5G64830
> AT5G64813
> AT5G61840
> AT5G48520
> AT5G46190
> AT5G45300
> AT5G41480
> AT5G38380
> AT5G38360
> AT5G37310
> AT5G24360
> AT5G18190
> AT5G11490
> AT5G11640
> AT5G11380
> AT5G07270
> AT5G05920
> AT5G05820
> AT5G04070
> AT5G02850
> AT3G56460
> AT3G55480
> AT3G53090
> AT3G52610
> AT3G51040
> AT3G51050
> AT3G49850
> AT3G47390
> AT3G42050
> AT4G39920
> AT4G38040
> AT4G38500
> AT4G38180
> AT4G35530
> AT4G34110
> AT4G31150
> AT4G26410
> AT4G11970
> AT4G00420
> AT3G28670
> AT3G27320
> AT3G17430
> AT3G17450
> AT1G53770
> AT3G17750
> AT3G20870
> AT3G29010
> AT3G04470
> AT3G05040
> AT3G04970
> AT3G33520
> AT3G05090; AT3G05080
> AT1G13320
> AT1G68980
> AT1G73840
> AT1G73970
> AT1G34550
> AT1G79810
> AT1G01910
> AT1G76340
> AT1G80950
> AT1G79975
> AT1G54390
> AT1G54150
> AT1G05500
> AT2G40410
> AT2G36240
> AT1G78810
> AT1G08960
> AT1G08750
> AT2G17390
> AT1G76940
> AT1G30970
> AT2G28390
> AT2G38950
> AT2G27600
> AT2G27110
> AT2G41350
> AT2G35100
> AT2G35360
> AT2G40980
> AT2G19280
> AT2G39910
> AT2G41960
>
>
> > On Fri, Oct 2, 2009 at 4:05 AM, Neil Graham <neil from arabidopsis.info>
> wrote:
> >
> >> Hi
> >>
> >> Have a look at:
> >>
> >> Czechowsli et al 2005. Genome-wide identification of superior reference
> >> genes for transcript normalisation in Arabidopsis. Plant Physiology.
> >> 139:5-17.
> >>
> >> These genes were identified by finding genes with a low %CV across the
> >> AtGenExpress developmental series.
> >>
> >> Thanks
> >>
> >> Neil Graham
> >>
> >> --------------------------------------
> >> http://arabidopsis.info
> >>
> >> Nottingham Arabidopsis Stock Centre
> >> Plant and Crop Sciences Division
> >> University of Nottingham
> >> Loughborough
> >> UK
> >> --------------------------------------
> >>
> >> Karen Browning wrote:
> >>
> >>> What would be a good set of housekeeping genes to use for "standards"
> for
> >>> comparing expression data in Arabidopsis?
> >>>
> >>> Anyone have a standard list of At gene numbers that they use, e.g.
> actin,
> >>> tubulin, etc.?
> >>>
> >>
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> >>
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> >
> >
> >
> > --
> > Eve Syrkin Wurtele, Professor
> > Bioinformatics and Computational Biology
> > 2624D Howe Hall, VRAC,
> > Iowa State University, Ames IA 50011, USA
> > metnetdb.org
> > metablast.org                      515-708-3232 (cell)
> >
> >
>
>
> --
> Eve Syrkin Wurtele, Professor
> Bioinformatics and Computational Biology
> 2624D Howe Hall, VRAC,
> Iowa State University, Ames IA 50011, USA
> metnetdb.org
> metablast.org                      515-708-3232 (cell)
> _______________________________________________
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