GeneCalling
In the field of genomics, GeneCalling is an open-platform mRNA transcriptional profiling technique.[1] The GeneCalling protocol measures levels of cDNA, which are correlated with gene expression levels of specific transcripts. Differences between gene expression in healthy tissues and disease or drug responsive tissues are examined and compared in this technology.[2] The technique has been applied to the study of human tissues[3] and plant tissues.[4]
Method
In the GeneCalling protocol, mRNAs are first isolated from a given sample and processed into fragments for analysis. This usually involves the synthesis and subdivision of double-stranded cDNAs from polyA RNA. Distinct sets of restriction enzymes can then be used to digest sets of the divided cDNAs and resulting fragments ligated to labelled adapters to be amplified by PCR. PCR products are then purified and subjected to gel electrophoresis on a mounted platform employing stationary laser excitation and a multi-colour charge-coupled device imaging system.[5] A fluorescent label at the 5' end of one of the PCR primers allows for visualization of the PCR fragments, and the cDNAs are subjected to several isolated and identical restriction digests to generate a merged profile based on peak height and variance.[6] The merged digestion profiles from the cDNA preparations are then compared to locate differentially expressed fragments (such as between normal tissue and diseased or drug responsive tissue); these profiles are compared by means of various internet-ready databases such as GeneScape.[7]
References
- ^ Kirst, M. E. (1 May 2005). "Identification and Characterization of Endoplasmic Reticulum-Associated Degradation Proteins Differentially Affected by Endoplasmic Reticulum Stress". Plant Physiology. 138 (1): 218–231. doi:10.1104/pp.105.060087. PMC 1104177.
- ^ Green, Cynthia D.; Simons, Jan Fredrik; Taillon, Bruce E.; Lewin, David A. (April 2001). "Open systems: panoramic views of gene expression". Journal of Immunological Methods. 250 (1–2): 67–79. doi:10.1016/S0022-1759(01)00306-4.
- ^ Gene Expression Analysis by Transcript Profiling Coupled to a Gene Database Query. Nature Biotechnology 17, 798-803: 1999
- ^ Expression Profiling of the Maize Flavonoid Pathway Genes Controlled by Estradiol-Inducible Transcription Factors CRC and P. The Plant Cell 12, 65-79: 2000
- ^ Klein, S; de Fougerolles, AR; Blaikie, P; Khan, L; Pepe, A; Green, CD; Koteliansky, V; Giancotti, FG (August 2002). "Alpha 5 beta 1 integrin activates an NF-kappa B-dependent program of gene expression important for angiogenesis and inflammation". Molecular and Cellular Biology. 22 (16): 5912–22. PMC 133962. PMID 12138201.
- ^ Shimkets, R.A. et al. Gene Expression Analysis by Transcript Profiling Coupled to a Gene Database Query. Nat. Biotechnology 17, 798-803: 1999
- ^ Grotewold, Erich (2003). Plant Functional Genomics. Springer Science & Business Media. p. 386. ISBN 9781592594139. Retrieved 17 October 2016.