miRNA profiling of bilateral rat hippocampal CA3 by deep sequencing

https://doi.org/10.1016/j.bbrc.2011.05.004Get rights and content

Abstract

MicroRNAs (miRNAs) have been demonstrated to be potent post-trascriptional modulators of protein expression. miRNA expression was profiled in the left and right dorsal hippocampal CA3 of mature rats by high-throughput deep sequencing. Among the sequenced and cross-mapped small RNAs, 88% belonged to the miRNAs annotated in the miRBase 15 database. Nearly half of the small RNAs belonged to the let-7 family miRNA. Seven percent of the sequenced small RNAs were not annotated in miRBase 15. Bioinformatic analysis of the unannotated small RNA sequences suggested seventeen novel miRNA candidates with relatively high expression levels (>100 tags per million). The left:right expression ratios were similar for all highly expressed miRNAs with less than 10% differences. These results provide a basic idea of the relative expression strengths of known and unknown miRNAs in the dorsal hippocampal CA3.

Highlights

► miRNA pofiling of male rat hippocampal CA3 was performed by deep-sequencing. ► About half of miRNAs belonged to the let-7 family. ► There were no distinct differences between left and right CA3 in miRNA expression. ► Unannotated short RNAs were observed with significant clone counts.

Introduction

MicroRNAs (miRNAs) are evolutionally preserved short (18–25 nucleotide) RNAs that bind to the 3′ untranslated regions (UTR) of mRNAs to interfere translation (for a review [1]). Since the original discovery in nematodes [2], several hundred miRNAs have been identified in rodents. Some miRNA are enriched in particular cell types in the nervous system. For example, in the murine stem cell-derived system, miR-124 and miR-128 are specifically expressed in neurons [3], whereas miR-23, miR-26 and miR-29 are preferentially expressed in glia during cell differentiation [4]. Interestingly, miRNA expression are shown critical in coordinating the left–right axis of the nervous system in nematodes [5].

In rodents, the left and right hippocampi are interconnected by massive glutamatergic projections from CA3 pyramidal cells and hilar cells. Functional and morphological characteristics of hippocampal CA3-CA1 synapses have been shown to be biased by the laterality of CA3 neurons [6], [7]. Such asymmetrical distribution of CA3-CA1 synapses has been suggested to be in part genetically programmed [8]. Screening of genes by cDNA microarrays suggested some genes are more abundantly expressed in one side of the hippocampus [9], [10], [11], yet genes that pronouncedly sculpt the left–right asymmetry of mammalian hippocampus have not been identified.

Recent advances of high-throughput sequencing allow quantitative assessment of transcribed RNAs. Expression levels of miRNAs can be measured by sequencing PCR-amplified, linker-tagged small RNA libraries [12], [13]. In this study, we investigated the miRNA expression profiles in both sides of the CA3 region by high-throughput (deep) sequencing.

Section snippets

Preparation of CA3 samples

RNA was purified from five week old male Long-Evans rats. For each rat, the brain was quickly removed from the skull after decapitation and dissected in half at the midline. The brain was then immediately cooled in ice-chilled Ringer solution. The left and right brains were cooled in different containers. After cooling for at least 3 min, the brain was taken out of the cooling solution and the hippocampus was gently isolated using spatulas. Coronal sections (400 μm thickness) were prepared using

Results

Three sets of left and right hippocampal CA3 RNA cDNA libraries were analyzed by deep sequencing. Between 9.8 and 10.8 million tags were read from each samples totaling to more than 60 million sequenced small RNAs. Of these sequenced small RNAs, 93.3 ± 1.1% was mapped to previously identified miRNAs (miRBase release 15, www.miRBase.org) [18] or other small RNA sequences using a cross-mapping procedure [15]. The coefficient of variation (C.V.) of normalized clone count for each miRNA was computed (

Discussion

Surprisingly high proportions of let-7 family miRNAs were found in our samples compared with previously published deep sequencing results in the mouse whole brain [19] or hippocampus [17]. The discrepancy between our results and others is not clear. One potential cause of the difference is the use of LNA oligonucleotide in our experiments to remove adapter dimers and lesser number of PCR cycles (12 vs. 20 [20], [21]) to construct the small RNA library. It is noted that more recent deep

Funding

This work was supported by KAKENHI grants (21650081 to HH and YS, 21700440 to YS; 20770145 to MK) and a Grant of the Innovative Cell Biology by Innovative Technology (Cell Innovation Program, to Yoshihide Hayashizaki) from the MEXT of Japan. YS is supported by the Special Postdoctoral Researcher Program, RIKEN.

Acknowledgments

We thank Ms. Aki Hosoya, Dr. Michiel J.L. de Hoon and Mr. Shiro Fukuda for technical supports and/or comments. We acknowledge to RIKEN GeNAS for small RNA sequence data production.

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