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USC Team to Use Patch-Clamp, RNA-seq to Study Gene Expression Variability in Single Neurons

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Building on a method that combines patch-clamp with single-cell transcriptome sequencing, researchers from the University of Southern California in Los Angeles are applying it to study gene expression variation in neurons.

The team was one of three to recently receive funding from the National Institutes of Health's Common Fund, through its Single Cell Analysis Program. The other two groups include a team led by the University of San Diego, which is using RNA-seq to create a 3D transcriptional map of the human brain (IS 11/27/2012), and a team led by the University of Pennsylvania to use single-cell transcriptome sequencing and functional genomics technology to study transcriptome variability in heart and brain cells (IS 12/4/2012).

Jim Knowles, a professor of psychiatry and behavioral sciences at USC's Keck School of Medicine, and Robert Chow, an associate professor of physiology and biophysics, also at the Keck School of Medicine, are heading up the USC-led team under a five-year $9 million grant from the NIH.

They are using a technique developed in Knowles' lab that combines patch-clamp with single-cell RNA-seq, which allows them to take electrophysiology measurements of cells and then sequence the cells' transcriptomes and thus correlate gene expression with phenotypes (IS 8/21/2012).

The goal of the NIH-funded project is to determine how much variability in transcriptomes of single cells from the same cell type is due to technical noise and how much is true biological variation.

The team plans to use the patch-clamp RNA-seq method on four different types of cells: fusion cells found in placenta known as syncytiotrophoblasts, olfactory neurons, Purkinje neurons, and cortical neurons.

These four cell types were chosen because the NIH funding announcement stipulated that the cells must be healthy, human cells from live, intact tissue. Collaborating with surgeons at USC, the team determined that these cells would be the most readily obtainable and easy to work with.

The four different types of cells will be obtained from patients who are already undergoing surgery. The researchers have obtained permission to collect the cells for research instead of discarding them. The Purkinje neurons, for instance, will be obtained from patients with severe epilepsy who are undergoing neurosurgery to prevent seizures, while the cortical neurons will be removed from patients having surgery to remove a blood clot or cancer.

"All four cells were chosen because they're easily procured for us, and in all cases, even though tissue is removed for some other purpose — say removing a cancer tumor or a blood clot — we can get some normal tissue without harming the patient," Chow told In Sequence.

The patch-clamp RNA-seq protocol involves first using a patch electrode to measure membrane and electrophysiological properties of a single cell, after which the cytoplasm can be sucked out for RNA-seq.

The original method, published in Frontiers in Genetics last year, used Clontech's SMARTer Ultra Low input RNA kit, but Knowles told In Sequence that since then the lab has tested NuGen's Ovation Ultralow kit as well as ScriptSeq, developed by Illumina's Epicentre.

One potential advantage of the NuGen kit is that "it might provide more linear results at the lower expressed transcripts," said Knowles. And the advantage of ScriptSeq is that "it is directional and [can sequence] more than just poly-A RNA." However, with ScriptSeq there is "a lot of variability across the same gene," he added.

Sequencing will be done on the Illumina HiSeq 2000 and the 2500, when the lab receives the upgrade for one of its machines.

Currently, Knowles said the team is in the process of setting up the lab space and purchasing equipment and that in the second half of the first year of the grant, the team will begin sequencing.

By the end of the first year, he anticipates as many as 1,000 cells will be sequenced. In total, at the end of five years, the team expects to sequence several thousand cells.

The team will be doing several different types of experiments to determine technical and biological variability between the cells.

The placental syncytiotrophoblasts were chosen because they are large fusion cells, so have more RNA than typical cells. This gives the researchers the ability to create two different RNA-seq libraries from the RNA in a single cell. Knowles said that the sequencing experiments from these cells will serve as a way to measure technical variability, since gene expression should be identical from an RNA-seq study of the same cell.

Typically, measuring the extent of technical variability in single-cell sequencing experiments is not feasible, since "once you use one cell for RNA-seq, you never have that cell again," Knowles said. But the larger, fusion placental cells contain just enough RNA to create two sequencing libraries.

The olfactory neurons were chosen because "the dogma at the moment is that each neuron expresses one olfactory receptor," said Knowles. So sequencing these cells also acts as a control, because each cell should be expressing a different receptor, he added.

Finally, the two different neural cells were chosen because gene expression is expected to vary from cell to cell, but it is unknown by how much.

With the neural cells, the team is also measuring gene expression in the context of a stimulus to see how gene expression of those cells changes before and after a stimulus.

For each cell type, the researchers will perform several different RNA-seq experiments to analyze biological noise. First, 10 cells from each type will be pooled in duplicate. One pool of 10 cells will be sequenced, the other pool will be divided into 10 samples and each sample will be sequenced separately. Additionally, 10 cells will each be sequenced separately.

The researchers chose to use the patch-clamp technique for isolating cells because not only does it enable electrophysiology measurements, but it is also a precise way to ensure that only one cell is selected without disrupting the cell itself.

The technique itself is more laborious than something like laser capture microscopy, Knowles said, but patch-clamp gives a "real phenotype of the cell."

"We can see its entire morphology and be certain we're in a single cell. With LCM, it's almost impossible to get just a single cell," he said. Additionally, it is difficult to perform LCM without damaging the cell, which has the potential for changing expression, he said.

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