What do you get when you cross eastern medicine with molecular biology?
How about a novel way to isolate mRNA from living cells using acupuncture?
That’s just one of many projects going on in Dr. Karl Hasenstein’s lab at the University of Louisiana, Lafayette.
Original research performed by Dr. Hasenstein’s lab and presented at the CHI qPCR conference earlier this year demonstrated glass needles coated with oligodT could successfully isolate mRNA direct from single cells (for example, Drosophila eggs) and allow for the ability to quantify gene distribution spatially across a gradient within a cell or over time in a developing cell.
The technique, called Solid Phase Gene Extraction, is clean, fast, and specific.
While this was a powerful technology, they noticed some issues which they contributed to using glass needles. The glass needles could not be consistenly coated on each tip so variation was significant and the glass had a tendency to kill the cells.
The solution: steel acupuncture needles.
The stainless steel needles provided uniformity of coating with oligodT, allowing for internal normalization in each capture, and the needles did not kill cells. Dr. Hasenstein reported that he could pierce a Drosophila egg 4 times to remove mRNA and the eggs still developed normally into adult flies.
What was really impressive is the kinetics of binding. Data presented showed that within 60 seconds, the amount of RNA captured in the vicinity of the needle reached full saturation.
To elute the captured mRNA, the needles are placed in a PCR tube with 5 µL of buffer and one-step qRT-PCR carried out on the complete captured material. qRT-PCR results demonstrated very sensitive and correct expression of RNA in the Drosophila embryo that correlated precisely with existing data for the genes examined.
Experiments with budding seeds and tumor tissues was also successful in quick mRNA capture without destroying the sample, a major advantage of the technique. Rare or limiting specimens can be examined for expression levels without harm so they can be used for other studies requiring DNA or protein analysis.
And in cases where the sample needs to be shared among many researchers, this can be an efficient way to collect as much data out of a single sample as possible.
Additional applications for this technique are easy to foresee. Forensic samples could be probed with stainless steel-coated STR-linked capture probes to pull human DNA out of very tiny and dilute samples and used directly in STR profiling, avoiding the need for nucleic acid extraction. Seeds could be sampled for genetic modifications without concern for PCR inhibitors. Antibody-coated needles can allow for quick screening of viral particles direct from patient serum or blood. The possibilities for increasing scientific discovery as well as the commercial potential are boundless.
To continue further studies Dr. Hasenstein explains that additional funding will be needed to support research efforts. They are looking for commercial partners to help develop the method for use in more than just Drosophila. Based on sensitivity of the qPCR results presented at the CHI conference, the potential uses appear to be well worth the investment.