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First published online June 6, 2005
Journal of Experimental Biology 208, 2389-2398 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01625

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Determination of the exact copy numbers of particular mRNAs in a single cell by quantitative real-time RT-PCR

Akiko Wagatsuma¹, Hisayo Sadamoto^1,2,3, Takashi Kitahashi⁴, Ken Lukowiak⁵, Akihisa Urano^1,4 and Etsuro Ito^1,2,*

¹ Division of Biological Sciences, Graduate School of Science, Hokkaido University, North 10, West 8, Kita-ku, Sapporo 060-0810, Japan
² Division of Innovative Research, Creative Research Initiative `Sousei' (CRIS), Hokkaido University, North 21, West 10, Kita-ku, Sapporo 001-0021, Japan
³ Laboratory of Functional Biology, Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, 1314-1 Shido, Sanuki 769-2193, Japan
⁴ Field Science Center for Northern Biosphere, Hokkaido University, North 9, West 8, Kita-ku, Sapporo 060-0809, Japan
⁵ Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada

View larger version (56K): [in a new window]   Fig. 1. Isolation of a single, identifiable neuron from the Lymnaea CNS. (A) Ventral view of the right cerebral ganglion in L. stagnalis. The arrow indicates the cerebral giant cell. Scale bar, 100 µm. (B) The same cerebral ganglion after isolation of the cerebral giant cell. (C) Light microscopic photograph of the isolated cerebral giant cell. Scale bar, 50 µm. (D) Hoechst staining of the same cell. (E) Merged image of (C) and (D). Only the nucleus of the isolated, single cerebral giant cell was observed, indicating that no other cells were removed along with the cerebral giant cell.

View larger version (17K): [in a new window]   Fig. 2. Assessment of qRT-PCR assay by a comparison of RT-PCR efficiencies between standard RNA solutions and sample mRNA solutions that contain cell debris. The solid circles show the relation between the starting quantity of RNA and the threshold cycles for standard Lymnaea CREB2 RNA solutions. The slope of the regression line is –3.98. The open circles show the relation between the starting quantity of mRNA and the threshold cycles for the Lymnaea CREB2 mRNA samples obtained from the cerebral giant cells. The amount of total mRNA in the sample, when 19 cells in a tube were used, was arbitrarily assigned a value of one. The slope of the regression line is –3.94. The two parallel slopes confirmed that RNAs of standard RNA solutions and samples could be amplified with the same RT-PCR efficiency in qRT-PCR.

View larger version (49K): [in a new window]   Fig. 3. Assessment of qRT-PCR assay by determination of the copy number of exogenous RNA at a known concentration in reaction solutions. (A) Amplification plots generated by the increase of reporter dye fluorescence with each cycle of PCR of salmon GTH 2 cDNA. The solid boxes show standard solutions, and the open boxes show the test samples. All reactions were performed in duplicate (1 µl each, which was 1/15 of the total volume of the reverse-transcribed products). An arbitrary threshold cycle was obtained in the exponential phase of PCR. (B) Standard curve for the starting quantity of salmon GTH 2 RNA. The solid circles show standard solutions, and the open circles the test samples. (C) Copy numbers of salmon GTH 2 RNA in 1 µl samples (1/15 of the total volume of the reverse-transcribed product). The total volume for reverse transcription was 15 µl with 6x104 RNA copies, and thus the detected value of 4143±71 copies (N=20 from the duplicates of 10 samples) in 1 µl of solution (i.e. 1/15 of the volume of the reverse-transcribed products) was reasonable. (D) Serial dilution of salmon GTH 2 RNA. The error at 10 copies shows that the S.E.M. is low enough (2.93, N=6 samples).

View larger version (56K): [in a new window]   Fig. 4. The copy numbers of Lymnaea CREB2 and CREB1 mRNAs in single cerebral giant cells. (A) Amplification plots generated by an increase of reporter dye fluorescence with each cycle of PCR of Lymnaea CREB2 cDNA. The filled squares show standard solutions, and the open squares show the test samples. All reactions were performed in duplicate (6 µl each, which was 6/15 of the total volume of the reverse-transcribed products). An arbitrary threshold cycle was obtained in the exponential phase of PCR. (B) Standard curve for the starting quantity of Lymnaea CREB2 RNA. The filled circles show standard solutions, and the open circles the test samples. The estimated copy number of Lymnaea CREB2 mRNA in a single cerebral giant cell was between 30 and 240. (C) Amplification plots generated by an increase of reporter dye fluorescence with each cycle of PCR of Lymnaea CREB1 cDNA. The notation is the same as (A). (D) Standard curve for the starting quantity of Lymnaea CREB1 RNA. The notation is the same as (B). The estimated copy number of Lymnaea CREB1 mRNA in a single cerebral giant cell was below the determination sensitivity.

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