The overall goal of this protocol is to investigate the association of aberrant splicing with drug resistance profiles in in vitro models of solid tumors and hematological malignancies. This method can help answer key question in the field of molecular pharmacology and chemotherapy resistance such as which are the mechanism underlying drug resistance on post-transcriptional level? The main advantage of this technique is that it combines well established cytotoxicity screening protocol with powerful next generation sequencing based transcriptomic analysis.
This technique could lead to a better understanding of the molecular mechanisms underlying chemotherapy failure and it will improve the early detection of drug resistance. This method can provide insight into genome-wide determinants of drug resistance in cell line models. But it can also be applied to other systems such as primary patient samples and xenograft.
Demonstrating this procedure will be Johan Fomirno, a technician from our laboratory at the Cancer Center Amsterdam. To perform the MTT assay for leukemic cells, prepare 96-well flat bottom experimental plate. Dedicate wells to drug concentrations to control cells and to control medium without cells.
Prepare a drug dilution range of dexamethasone or dex. Add 30 microliters from each dex dilution into an appropriate well of the 96-well plate. Make sure to include each concentration in triplicate.
Harvest exponentially growing leukemic cells and re-suspend them at their optimal seeding concentration. Add 120 microliters of the cell suspension to each well containing either the drug solution or the growth medium, and incubate at 37 degrees Celsius with 5%carbon dioxide for 72 hours. After the incubation step, add 15 microliters of MTT reagent to each well by using a repetitive pipette.
Shake the plate for five minutes with a plate shaker up to a maximum of 900 shakes per minute. Place the plates back at 37 degrees Celsius with 5%carbon dioxide in a cell culture incubator and incubate for another four to six hours. Following incubation, make sure to thoroughly re-suspend the formazan crystals as the absorbance values depend on proper solubilization of the compound.
Add 150 microliters of the acidified isopropanol to each well and mix well with a multichannel pipette to thoroughly re-suspend all the formazan crystals. Using a microplate reader, determine the optical density or OD at 540 and 720 nanometers to ensure an accurate measurement by correcting for background OD.To perform SRB assay for pancreatic carcinoma cells, prepare a 96-well flat bottom experimental plate. Seed pancreatic carcinoma cells growing in exponential phase in triplicate in a 96-well flat bottom plates at the appropriate density in 100 microliters of medium by using a multichannel pipette.
Add 100 microliters of medium to medium-only wells and incubate overnight at 37 degrees Celsius with 5%carbon dioxide to ensure proper adhesion of the cells to the plate. Add 100 microliters from each dilution into an appropriate well of the 96-well plate by using a multichannel pipette. Make sure to have each concentration in triplicate.
Incubate at 37 degrees Celsius with 5%carbon dioxide for 72 hours. Next, add 25 microliters of cold TCA solution to the wells by using a multichannel pipette. Incubate the plates for at least 60 minutes at four degrees Celsius to precipitate and fix the proteins at the bottom of the wells.
Empty the plate by removing the medium and dry briefly on a tissue. Wash five times with tap water before emptying the plate and drying at room temperature. Add 50 microliters of SRB solution per well by using a repetitive pipette and stain for 15 minutes at room temperature.
Then, empty the plate by removing the SRB stain. After washing the plate four times with 1%acetic acid, empty the plate and let it dry at room temperature. Add 150 microliters of Tris solution per well by using a multichannel pipette and to mix for three minutes on a plate shaker up to a maximum of 900 shakes per minute.
Read the optical density at 540 nanometers. Spin down the samples at 300 times g for three minutes. After removing the supernatant, extract total mRNA using commercially available silica membrane spin columns.
Determine the concentration and purity of total RNA by using UV-Vis spectrophotometer. To prepare the sequencing library, use two micrograms of total mRNA per sample. Follow mRNA library preparation protocol according to manufacturer's instructions.
After pooling the libraries in a single sample up to a final concentration of 10 nanomoles per liter using a bioanalyzer, use a high throughput sequencing system with single read 100 base pair mode. Design all primers as described in the text protocol. For first strand cDNA synthesis, set up reverse transcription of one microgram of the isolated RNA to cDNA by using 200 units per microliter of Moloney murine leukemia virus reverse transcriptase in its reaction buffer diluted one to five with sterile water.
To perform the RT-PCR reaction, add one microliter of cDNA to the PCR reaction mix and place the tubes in the thermocycler. Run the program as described in the text protocol. Following PCR, load the samples into a 1%agarose gel and run the gel in TBE buffer at 100 volts for approximately 30 minutes in an electrophoresis system.
To avoid cross contamination, use filter tips while performing the qRT-PCR assay. To perform qRT-PCR, prepare cyanine green PCR reaction mix for each specific splice variant to be detected as well as for the housekeeping gene. Load the master mix on a white 96-well plate and prepare duplicates for each sample.
Add five microliters of cDNA diluted 10 times in water to each mix before placing the plate in the thermocycler. Growth inhibition curves as determined by MTT assay for leukemic cells and SRB assay for pancreatic carcinoma cells are depicted here. Curves for dexamethasone resistant and gemcitabine resistant cells are shifted to the right indicating growth inhibition as compared to the parental sensitive cells.
The concentration and quality of cDNA libraries for all cell lines is determined with a bioanalyzer DNA chip. Electropherograms show fluorescence peaks at approximately 300 base pairs which are indicative of good quality for the subsequent RNA sequencing procedure. Short reads of the gene candidate DDX5 can be visualized as Sashimi plots where connecting lines represent splice junctions.
DDX5 transcript shows differential usage of exon 12 and CEM wild-type and CEM/R30dm cells as compared to glucocorticoid-resistant cell lines, CEM-C3 and CEM-R5. RNA sequencing results are validated by performing RT-PCR on selected genes for which primer pairs and yield to exons located upstream and downstream of the alternatively spliced exon. Differential splicing of the DDX5 gene is shown here.
Quantification of differentially-spliced transcripts through qRT-PCR assay using isoform-specific reverse primers confirms the specific down regulation of the DDX5 delta exon 12 splice variant and a specific glucocorticoid-resistant sub-lines. Once mastered, this technique can be done in three to four weeks. While attempting this procedure, it's important to remember to keep RNA samples on ice in order to avoid degradation.
Following this procedure, other methods like ectopic overexpression of novel splice variant can be performed. This will answer additional questions like functional impact of alternate splicing profile on drug resistance. This technique can pave the way for researches in the field of molecular pharmacology to explore genome-wide determinants of drug resistance.
