Isolating mRNA using acupuncture (BioTechniques Weekly)
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 the work Karl Hasenstein, Ph.D., presented at the CHI qPCR conference held in San Diego this past week.
Original research performed by Dr. Hasenstein’s lab demonstrated glass needles coated with oligodT could successfully isolate mRNA from single cells (for example, Drosophila eggs) and increasing 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. The only problem was that with glass needles, there was too much variation in the amount of coating on each tip, and that the glass had a tendency to kill the cells. So they switched to steel acupuncture needles. The advantages of stainless steel needles are the uniformity of coating, allowing for internal normalization in each capture, and the needles do not kill cells. 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.
The needles were then placed in a PCR tube and mRNA eluted 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. Additional work used the same technique for budding seeds and for examining tumor tissues.
The power of the acupuncture technique is that rare or limiting specimens can be examined for expression levels without harming them so they can be used for other studies such as DNA or protein analysis.
In cases where the sample needs to be shared among many researchers, this can be an effcient 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 and avoiding the need for nucleic acid extraction. Seeds could be sampled for genetic modifications without concern for having to accomodate difficult PCR inhibitors. Antibody-coated needles can allow for quick screening of viral particles direct from patient serum or blood without tedious procedures. The possibilities for increasing scientific discovery as well as the commercial potential are boundless.
To continue further studies 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.