Our protocol utilized RNA-Seq and bioinformatical tools to quickly uncover the overexpressed driver genes that contribute to the formation of stem-like colorectal tumorspheres. This protocol is used to screen a differentially expressed gene and signal pathways to investigate a molecular mechanism for cancer stemness in a selective colorectal cancers. On day seven of culture, use an inverted microscope with a digital cell imaging system to observe and measure the diameter of the tumorspheres.
When the tumorspheres reach a greater than 100 micrometer diameter, treat the culture with 0.25%trypsin. After five minutes at 37 degrees Celsius, neutralize the trypsin with two times the volume of growth medium and count the cells using a hemocytometer. Collect the cells by centrifugation.
And resuspend the pellet at a five times 10 to the four cells per 100 microliters of medium concentration. Add two microliters of the primary antibody of interest to each tube and incubate the cells for 30 minutes at room temperature and 200 revolutions per minute. At the end of the incubation, wash each tube with 900 microliters of PBS and analyze the cells for expression of the protein of interest by flow cytometry according to standard protocols.
For RNA isolation after trypsin collection as just demonstrated, resuspend the cells at a two times 10 to the fifth cells per 50 microliters of PBS concentration and lyse each sample with 200 microliters of lysis buffer supplemented with two microliters of beta mercaptoethanol per tube. After brief vortexing and a five minute incubation at room temperature, collect the lysates by centrifugation and add 200 microliters of 70%ethanol to each tube. Transfer the samples into rapid columns and remove the solvent by centrifugation.
Wash the samples with 400 microliters of wash solution one and 600 microliters of wash solution two to completely remove any non-RNA by centrifugation, followed by an additional two minutes centrifugation under the same centrifuge conditions to remove any residual ethanol. Then resuspend the pellets in 50 microliters of distilled water per tube and centrifuge the sample for one minute before collecting the RNA containing supernatants. To investigate the differential gene expression between tumorsphere cells compared to parental tumor cells after RNA sequencing analysis.
Select genes with a greater than one to minus one log2 fold change with read counts greater than 100 in the tumorsphere and parental groups. And use the commands as indicated. To obtain a volcano plot of the data to determine the differential gene expression.
For drive gene selection in network analyst, select single gene input and copy and paste the selected overexpressed genes with human specified as the organism and official gene symbol as the ID type. Click Upload and proceed. To insert and analyze the data using protein-protein interaction, following genetic protein-protein interaction, use the STRING interactome database with a confidence score cut off of 900 to show the seed genes.
Cross-linking the uploaded genes. The seed genes associating with more individual genes can be selected as driver genes that may be involved in maintaining formation of the tumorsphere. Select Proceed in the mapping overview white in the Background and Force Atlas in the Layout knob.
Then select PANTHER base pair to analyze the upregulation gene group. Tumorspheres of greater than 100 micrometers in diameter form within seven days of culture. Flow cytometric analysis reveals an increase in LGR5 and CD133 expression in HT29-derived tumorspheres compared to parental HT29 cells.
After RNA sequencing, the genes with a greater than one log2 fold change in their up or downregulation with a P-value less than 0.05 as determined by heat map can be plotted to distinguish the significant genes between HT29-derived tumorspheres and parental HT29 cells. In this representative experiment, analysis of the upregulated genes with a greater than one log2 fold resulted in 10 seed genes, cross-linking the gene networks. PANTHER base pair analysis indicated that two genes were associated with negative regulation of apoptosis.
Moreover, the selected genes were consequently validated by quantitative PCR confirming their increased expression in HT29-derived tumorspheres. The differential gene selection is the most important step of the procedures. Remember to use a log2 fold change greater than one with recounts greater than 100 as the criteria.
Using this protocol, the genes selected from the colorectal-derived tumorspheres can be knocked down or overexpressed to uncover their potential roles in tumorsphere formation.
