Researchers have developed a novel approach for studying post-transcriptional regulation in a smaller quantity of cells than before by using affinity capture beads. The new immunoprecipitation technique, called TrIP-chip, has shown to be as efficacious as traditional sucrose gradient centrifugation approaches, but with the added potential for clinical application.
Jingfang Ju, co-director and assistant professor of the Translational Research Laboratory at the Stony Brook University Medical Center, says TrIP-chip attempts to overcome the limitations of previous studies of actively translating messenger RNAs.
Ju says that, to date, most researchers have depended on the sucrose gradient ultracentrifugation approach to isolate mRNAs from polysomes. This method requires up to a billion cells, he says, virtually eradicating the potential for use in the clinical setting, where specimen cell count is generally limited.
The team, which represents a collaboration of researchers from the Mitchell Cancer Institute in Mobile, Ala., the Yale University School of Medicine, and the Columbia University Genome Center, in addition to Stony Brook, optimized an approach using chaperone protein hsp70 affinity capture beads to isolate actively translating mRNAs from as few as 500 cells.
They have demonstrated the success of the technique in vivo by analyzing post-transcriptionally regulated gene products obtained from HCT116, a human metastatic colon cancer cell line, and have published their results in Nucleic Acids Research.
"We did a comparison with a sucrose-gradient approach just to ensure that the new [TrIP-chip] approach actually captured most of the translated transcripts with a 5-fluorouracil treatment and we did achieve a great deal of overlapping between the two," Ju says.
5-fluorouracil, or 5-FU, is one of the most widely used chemotherapeutic compounds for the treatment of solid tumors.
"Because we believe that post-transcriptional and translational control plays an important role for the drug response and resistance mechanisms, we'd like to discover genes that are actually involved, post-transcriptionally, with 5-FU exposure," Ju says. The team hopes to identify additional gene targets associated with 5-FU resistance using this new method.
The next step for the scientists is to develop what they are calling a "TrIP-seq" approach by combining their TrIP-chip methods with a next-generation sequencing platform. Ju says that in the future they intend to "increase the sensitivity of this approach," and that "hopefully we can perform these within a single cell."