Abstract
Standard procedures for transcriptome analysis usually requires more than one microgram of total RNA. For the HiCEP analysis, we first needed 1~2μg of polyA RNA (corresponded to 100μg of total RNA). However, actual transcriptome investigation often requires a new protocol which allows us to carry out analysis using less than 10,000 cells (corresponds to 0.1μg of total RNA). Here, therefore, we attempted to develop a new HiCEP way for a small number of cells.
First we carried out our examination using diluted samples of high quality RNA fraction. We amplified HiCEP templates which are products of HiCEP reaction and have adaptors at either end, by PCR with primers designed in the adaptors. By this amplification, quantity of total RNA required for the analysis decreased to 0.1μg(corresponds to ~10,000cells), allowing us to conduct HiCEP analysis on wide range of applications, diagnosis with blood, small material obtained by biopsy and so on. Subsequently, we optimized the conditions in several reaction steps and succeeded in HiCEP analysis using 0.5 ng total RNA, which corresponds to 50 mammalian cells.
Next, we used cells instead of the diluted RNA fractions as starting materials, namely we started HiCEP analysis with a small number of cells, which were collected under a microscope. Thus we obtained reproducible result of 10-cells analysis. Currently, we are focusing on single-cell analysis.
