Utilizing the GTEx Gene V8 and GNF Atlas Tracks in the UCSC Genome Browser to Evaluate PIR Expression

This week I will once again be using the UCSC Genome Browser (http://genome.ucsc.edu), this time to gather data on how PIR expresses the pirin protein (Kent, Sugnet, Furey, et al., 2002). One of the traditional methods of gene expression analysis is through the use of DNA microarray assays, a process in which a sample of mRNA is essentially reversed engineered into DNA through a Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). RT-PCR synthesizes DNA by taking the mRNA sample and utilizing an enzyme known as reverse-transcriptase as well as a polythymine primer (mRNA has a polyadenine tail) to create a single stranded complementary DNA molecule to the mRNA sample by deducing the proper nitrogenous bases for the DNA strand given the nitrogenous bases of the mRNA. This single stranded complementary DNA (cDNA) can then be dyed and placed into the microarray with any number of single stranded genes and DNA polymerase, thereby allowing the gene DNA to bind to the cDNA (Unbound cDNA is flushed out of the array). If cDNA binds with a gene, the segment of the array where that gene is located will appear to be the color that the cDNA was dyed, and it can be inferred that this gene is expressed in the tissue that the mRNA used to create the cDNA was derived from, because the cDNA was derived from the sample mRNA which in turn was templated from the type of gene that the cDNA was compatible with (Farina, 2020). Though modern science has produced technologies capable of simply reading RNA in a sample through a process known as RNA sequencing (Shortened to RNA-Seq) in which technologies known as Next Generation Sequencers are capable of performing millions (or even billions) of readings, understanding microarray assays has helped me to better grasp the process of mRNA transcription. 

Navigating to the Genome Browser assembly for PIR, I turn on the tracks for GTEx Gene V8 (Analyzes gene variations and expression in different human tissues) and GNF Atlas (Analyzes gene variations and expression under different biological conditions) (Madeira, Park, Lee, et al., 2019). Clicking on the GTEx track displays information on pirin gathered by The GTEx Project (GTEx stands for NIH Genotype-Tissue Expression). The project has gathered 17,382 tissue samples from 948 individuals and has concluded that pirin is expressed in median levels in 52 types of human tissue. The GTEx Project is capable of gathering this data by utilizing the hg38/GRCh38 genome to align the genetic information of tissue samples after introns are spliced out and areas of exon overlap between genes are filtered from the sample data (Isoform collapse). The resulting DNA samples can then be aligned with corresponding DNA from the known hg38/GRCh38 genome to analyze the median levels of expression of a given gene within a given tissue without having to remap the genome of each individual tissue sample. The GTEx Project accomplishes this through the use of STAR v2.5.3a, a RNA-Seq program capable of reading alignments, as well as through the implementation of RNA-SeQC tool (v1.1.9), a program designed to filter and optimally parse genetic data to ensure that high quality data analysis is possible. At least, I think that is how it works; I am not fully confident that I understood The GTEx Project’s methodology (Though it does make a good topic for potential future research). To control the reliability of the data collected, tissue samples were inspected by pathologists to ensure the absence of genetic diseases and disorders (Kent, Sugnet, Furey, et al., 2002).

The GTEx Gene V8 track provided by the genome project is attached below. However, as blogger has had a nasty habit of downgrading the resolutions of my uploaded images, I will be attaching a link to the UCSC Genome Browser that will display the full PIR assembly for the GTEx Gene V8 and GNF Atlas tracks that I utilized during this week’s research at the end of this post. 

After exploring the GTEx Gene V8 track, I navigate to the GNF Atlas track. GNF Atlas is a public database from the Genomics Institute of the Novartis Research Foundation that seeks to catalogue how genes are expressed in tissues subjected to a variety of biological conditions. According to the Genome Browser’s description of the GNF Atlas track: “[The track] contains two replicates each of 79 human tissues run over Affymetrix microarrays.” This methodology contrasts with the GTEx Gene V8 track’s use of NGS technologies. In line with the previous weeks of research conducted on the PIR gene and pirin, I find through the GNF Atlas track that PIR is most highly expressed in cancerous tissues. However, even in cancerous tissues, PIR is still relatively underexpressed, something that may be important to keep in mind for future research (Kent, Sugnet, Furey, et al., 2002).

Given my current understanding of microarray assays and RNA-Seq technologies, it seems that the NGS technologies are far more efficient and thorough if utilized with properly implemented sequencing and parsing programs. However, from the outside looking in, many of these NGS technologies look to be prohibitively expensive. Furthermore, the programs used to conduct and analyze RNA-Seq processes appear to be extremely technical, and likely require a significant amount of training and experience to properly operate. While I would like to use NGS technologies one day, I think it would be best for me to first familiarize myself with microarray assays before moving on to more advanced sequencing techniques. 

Link to the Genome Browser assembly for PIR used in this post: http://genome.ucsc.edu/cgi-bin/hgTracks?db=hg38&lastVirtModeType=default&lastVirtModeExtraState=&virtModeType=default&virtMode=0&nonVirtPosition=&position=chrX%3A15384799%2D15493564&hgsid=1224333939_xvS8UPcGYDesq85TjMv3ZmzW4ERK

References

Farina D. 2020 Apr 15. Gene Expression Analysis and DNA Microarray Assays
[Internet]. YouTube.com; [cited 2021 Nov 20]. Available from:
https://www.youtube.com/watch?v=Hv5flUOsE0s

Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler D.  The
human genome browser at UCSC. Genome Res. 2002 Jun;12(6):996-1006.

Madeira F., Park Y.M., Lee J., Buso N., Gur T., Madhusoodanan N., Basutkar P., Tivey
ARN., Potter SC., Finn RD., Lopez R. (2019) The EMBL-EBI search and
sequence analysis tools APIs in 2019 Nucleic Acids Research, April 12, 2019;
doi: 10.1093/nar/gkz268