Description
The Gencode Intron Validation track shows
gene structure validations generated by the
GENCODE project.
This track serves as a companion to the Gencode Genes track.
The items in this track are colored based on the validation status determined
via RT-PCR of exons flanking the intron:
| Status |
Color |
Validation Result |
| RT_positive |
green |
Intron validated (RT-PCR product corresponds to expected junction) |
| RACE_validated |
green |
Intron validated (RACE product corresponds to expected junction) |
| RT_negative
| red |
Intron not validated (no RT-PCR product was obtained) |
| RT_wrong_junction |
gold |
Intron not validated, but another junction exists between the two
(RT-PCR product does not correspond to the expected junction) |
Methods
Selected gene models from the Genecode Genes track were picked for RT-PCR
and RACE verification experiments.
RT-PCR and RACE experiments were performed on the objects, using a variety of
human tissues, to confirm their structure. Human cDNAs from 24 different
tissues (brain, heart, kidney, spleen, liver, colon, small intestine,
muscle, lung, stomach, testis, placenta, skin, peripheral blood
leucocytes, bone marrow, fetal brain, fetal liver, fetal kidney, fetal
heart, fetal lung, thymus, pancreas, mammary gland, prostate) were
synthesized using twelve poly(A)+ RNAs from Origene, eight from Clemente
Associates/Quantum Magnetics and four from BD Biosciences as described in
[Reymond et al., 2002a,b]. The relative amount of each cDNA was
normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by quantitative
PCR using SyberGreen as intercalator and
an ABI Prism 7700 Sequence Detection System.
Predictions of human genes junctions were assayed experimentally by
RT-PCR as previously described and modified [Reymond, 2002b;
Mouse Genome Sequencing Consortium, 2002; Guigo, 2003].
Similar amounts of Homo
sapiens cDNAs were mixed with JumpStart REDTaq ReadyMix (Sigma) and 4
ng/ul primers (Sigma-Genosys) with a BioMek 2000 robot (Beckman). The
ten first cycles of PCR amplification were performed with a touchdown
annealing temperatures decreasing from 60 to 50°C; annealing
temperature of the next 30 cycles was carried out at 50°C. Amplimers
were separated on "Ready to Run" precast gels (Pharmacia) and
sequenced. RACE experiments were performed with the BD SMART RACE cDNA
Amplification Kit following the manufacturer instructions (BD
Biosciences).
Credits
Click here for a complete list of people who participated in the
GENCODE project.
References
Ashurst, J.L. et al.
The Vertebrate Genome Annotation (Vega) database.
Nucleic Acids Res 33 (Database Issue), D459-65
(2005).
Guigo, R. et al.
Comparison of mouse and human genomes followed by experimental
verification yields an estimated 1,019 additional genes.
Proc Natl Acad Sci U S A 100(3), 1140-5 (2003).
Mouse Genome Sequencing Consortium.
Initial sequencing and comparative analysis of the mouse
genome. Nature 420(6915), 520-62 (2002).
Reymond, A. et al.
Human chromosome 21 gene expression atlas in the mouse.
Nature 420(6915), 582-6 (2002).
Reymond, A. et al.
Nineteen additional unpredicted transcripts from human
chromosome 21. Genomics 79(6), 824-32 (2002).
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