Gene expression was analyzed in individual cells

Swedish scientists have developed a technique that allows to isolate individual cells from a tissue sample and analyze gene expression in them. The results of the work are published in the journal Nature Communications (Nichterwitz et al., Laser capture microscopy coupled with Smart-seq2 for precise spatial transcriptomic profiling).

The staff of the Karolinska Institute used a combination of two methods: laser entrapping microdissection (LCM, LCM) and full transcriptome analysis using Smart-seq2 RNA sequencing technology. The principle of LZM is that the cell of interest is selected using a microscope in a tissue sample, after which the sample is covered with a transparent film and the cell of interest is "glued" to it with a laser without destroying it. Smart-seq2 allows synthesizing full-size complementary DNA (cDNA) based on matrix RNA (mRNA) of a single cell, which then amplified by PCR and sequenced.

The combination of LZM with transcriptome analysis has been used before, but such techniques required from 200 to 4000 cells to obtain a sufficient amount of RNA for analysis. However, in some cases it is necessary to evaluate the difference in gene expression between individual similar cells in a small tissue sample. The use of Smart-seq2 and several improvements of the combined technique allowed scientists to evaluate the transcriptome of single cells with sufficient accuracy.

During the work, the researchers prepared 12-micrometer sections of the spinal cord tissue of newborn mice and visualized motor neurons in them with a special dye. At the same time, they successfully used not a standard expensive set of reagents, but fixed the cells with ordinary dehydrated ethanol. Isolated individual cells with LZM were subjected to direct lysis with hypotonic solution and Smart-seq2 was performed without extraction and purification of RNA required for standard sequencing techniques.

The scheme of LCM-seq (from an article in Nature Communications)

The experiment started with 120 neurons, gradually reducing their number to 50, 30, 10, 5, 2 and 1 cells. It turned out that the cDNA yield using direct lysis of 50, 30 and 10 cells is comparable to the results of RNA extraction and even allows more genes to be isolated from a sample unit on average (according to researchers, this is due to the loss of part of the genetic material during RNA purification). Moreover, the new technique, called LCM-seq, provided a success rate of full transcriptome analysis of 62 percent for one cell, 81 percent for two cells and 100 percent for samples containing five or more cells.

The effectiveness of LCM-seq was confirmed by revealing with its help a difference in gene expression in motor neurons similar in structure and function from different parts of the mouse spinal cord. After that, using the technique, the scientists successfully determined the difference in transcriptomes of similar motor neurons from postmortem samples of the spinal cord of patients with amyotrophic lateral sclerosis, as well as the substantia nigra and ventral peritoneal region of the brain of patients with Parkinson's disease.

Thus, LCM-seq is suitable for analyzing gene expression in individual cells, even in extremely small or partially decomposed tissue samples, the researchers write. One of them, Qiaolin Deng, noted that it is simpler, cheaper and has much greater sensitivity and reproducibility than existing techniques. According to scientists, their development can find wide application in various fields of biology and medicine, including in the study of cancer development and identification of its biomarkers.

Portal "Eternal youth" http://vechnayamolodost.ru  12.07.2016