Differential Display is a powerful technology that can be used to visualize differences in gene expression between or among different cells or tissues. This technology works by the systematic amplification of the 3' terminal portions of mRNAs and the resolution of those fragments on a DNA sequencing gel.
The RNAimage Kit from Genhunter is designed to simplify the process of Differential Display. It uses three one-base anchored oligo-dT primers (designed to bind to the 5' boundary of the poly-A tails) that contain 5' HindIII sites. These primers are used in combination with a series of arbitrary 13mers (also containing 5' HindIII sites) to reverse transcribe and amplify (using radiolabeled nucleotides) the mRNAs present in a sample of total RNA. Because the 3' untranslated regions of mRNA are AT rich, radioactively labeled dATP is used which greatly increases the specific activity of the labeled PCR product. The mRNA subpopulations are visualized by denaturing polyacrylamide gel electrophoresis. This allows the direct, side-by-side comparison of most of the mRNAs in two different cell populations.
The components of this kit have been well thought-out. The one-base anchored primers minimize the redundancy of the displayed cDNAs and simplify the reverse transcription reactions. The optimal length of arbitrary primers has been determined by statistical consideration, ensuring that each primer will recognize 50-100 mRNA species, which guarantees that most of the mRNA in the sample can be represented. Based on a theoretical calculation, 80 different random 13mers, divided in 10 kits, are sufficient for covering about 96% of 10,000 expressed mRNA species in a mammalian cell. The HindIII sites on the primers facilitate the manipulation of the cDNA after cloning. This is necessary because after you have run the gel with the radioactive PCR products, and it is dried and exposed to film, the identified differentially expressed bands are cut away from the gel and the DNA is recovered and amplified by PCR.
The company provides kits for early and late steps of the differential display protocol. This includes a cleaning system for the removal of genomic DNA after total RNA extraction, a cloning system for the differentially expressed PCR product isolated from the gel, and a kit for “Reverse Northerns” to identify false positives.
The results from this technique are highly reproducible and the RNAimage Kit is extremely efficient in amplifying many different mRNAs. The striking sensitivity of Differential Display allows you to detect mRNAs present in very small amounts. There is no doubt about the reliability of the RNAimage system and of the all kits provided.
That is not to say that there aren’t some minor problems with this system. For instance, for obvious reasons, it is extremely important to remove all genomic DNA when isolating total RNA. Unfortunately, long DNase I digestions potentially degrade mRNA, possibly affecting your results. Also, the GenHunter cloning vector contains three copies of the restriction site that is already flanking both sides of the PCR product you need to clone. This can make it hard to identify clones with insert after a restriction digest.
In summary, I would say that if you were planning on doing a differential screen, the RNAimage Kit is an excellent approach. The performance is outstanding and the system is really reliable because it rarely requires repetition and it generally gives clear results. I would highly recommend it to labs that need an excellent and reliable tool for the accurate screening of differentially expressed genes.
Luca Carta, Ph.D.
Mount Sinai School of Medicine
New York, New York