Daniel:Notebook/RNAFISH/2014-12-5

From ZhangLabWiki
Jump to navigation Jump to search

Cell Line Analysis[edit]

Back to Calendar

I've been using three cell lines to test the RNA FISH, U-87MG, T98-G, and BE2-C. None of the results from the 11/22/2014 test were conclusive, so I also got some data from Dr. Zhang on NCBI's website. This data is meant to see whether or not we have gene expression of the 4 genes I have already (RBFOX3/NeuN, FOXP2, CUX2, and GAD1) in the 4 cell lines, since we just assumed before. Links to the raw data can be found on the cell line pages (links above).

Gene Expression Analysis[edit]

I found two separate types of gene expression data sets. Two data sets (T98-G and U87-MG) were analyzed using Affymetrix microarrays, and one cell line (BE2-C) was analyzed using RNA-Seq.

Affymetrix Microarray Data (T98-G and U87-MG)[edit]

The first challenge of this is to understand the Affymetrix labeling system. With help from DAVID I changed the Affymetrix notation to Entrez Genes and identified which labels (usually several) corresponded to my genes of interest. The data is summarized below.

Gene Entrez Gene ID Gene List U87-MG Values T98-G Values
RBFOX3 146713 229406_at 1.654966 1.756282
RBFOX3 146713 235468_at 2.087149 2.173624
RBFOX3 146713 240975_x_at 3.09884 3.058095
FOXP2 93986 1562139_a_at 2.320036 2.509934
FOXP2 93986 1555516_at 1.644757 1.88334
FOXP2 93986 243278_at 1.420676 2.523345
FOXP2 93986 1552902_a_at 1.915386 2.145663
FOXP2 93986 235201_at 2.150443 3.007517
FOXP2 93986 1555647_a_at 2.238783 2.606263
FOXP2 93986 1555648_at 1.967491 2.01729
FOXP2 93986 1564876_s_at 3.461954 3.740007
FOXP2 93986 1555352_at 1.765198 2.079463
CUX2 23316 1566528_at 2.388545 2.225919
CUX2 23316 213920_at 1.672316 1.586615
CUX2 23316 1566527_at 3.108661 2.981597
GAD1 2571 205278_at 4.696471 1.507815
GAD1 2571 206669_at 4.935924 3.5153
GAD1 2571 206670_s_at 4.498899 2.625633

Since this is microarray data, the numbers don't mean a lot on their own (they range from about 1 to 14). So I plotted, for each cell line, the total distribution (boxplot) and also the individual values for each of the four genes.

So the U87-MG line might have an enrichment of GAD1, since that is the only gene for either line that appears to be significantly above the mean.

RNASeq Data (BE2-C)[edit]

This data is a little more tame. Basically I just sorted through a large text file until I found the chromosomal locations of each gene (using Gene Cards), then found the transcript name and FPKM. The table of results is below.

Gene Gene ID FPKM Orientation Confirmed
RBFOX3 ENST00000338834.4 0 - No
FOXP2 ENST00000393500.3 0.0004195009 + No
CUX2 ENST00000261726.5 0.6117341499 + Maybe
GAD1 ENST00000358196.3 0 + No

This one shows that the BE2-C line may contain appreciable amounts of CUX2. I'm basing this off the gene card for CUX2, which has the following information:

File:CUX2 GeneExpression.png

Using the same calculation for this (100*FPKM)^1/2, this would yield a score of 7.8, which for CUX2 seems to be about as high as it goes.