## 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 (PCR product corresponds to expected junction)
RT_negative | red | Intron not validated (no PCR product was obtained)
RT_wrong_junction | gold | Intron not validated, but another junction exists between the two (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).