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. 2005 Sep 28;33(17):5394-403.
doi: 10.1093/nar/gki863. Print 2005.

Real-time expression profiling of microRNA precursors in human cancer cell lines

Jinmai Jiang  1 , Eun Joo Lee, Yuriy Gusev, Thomas D Schmittgen
Affiliations

Real-time expression profiling of microRNA precursors in human cancer cell lines

Jinmai Jiang et al. Nucleic Acids Res. .

Abstract

Our previous study described a real-time PCR method to quantify microRNA (miRNA) precursors using SYBR green detection [T. D. Schmittgen, J. Jiang, Q. Liu and L. Yang (2004) Nucleic Acids Res., 32, e43]. The present study adapted the assay to a 384-well format and expanded it to include primers to 222 human miRNA precursors. TaqMan minor groove binder probes were used to discriminate nearly identical members of the let-7 family of miRNA isoforms. The miRNA precursor expression was profiled in 32 human cell lines from lung, breast, colorectal, hematologic, prostate, pancreatic, and head and neck cancers. Some miRNA precursors were expressed at similar levels in many of the cell lines, while others were differentially expressed. Clustering analysis of the miRNA precursor expression data revealed that most of the cell lines clustered into their respective tissues from which each cell line was ostensibly derived. miRNA precursor expression by PCR paralleled the mature miRNA expression by northern blotting for most of the conditions studied. Our study provides PCR primer sequences to all of the known human miRNA precursors as of December 2004 and provides a database of the miRNA precursor expression in many commonly used human cancer cell lines.

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Figures

Figure 1
Figure 1
Primer and TaqMan probe sequences to let-7 miRNA isoforms. The sequences of the miRNA precursors for the members of the human let-7 family of miRNA isoforms are shown. Underlined, sequence of the mature miRNA; red, sequences of the forward PCR primers; blue, sequences of the reverse PCR primers; green sequences of the TaqMan MGB probe; and yellow, priming sequences that differ among isoforms. Sequences are in the 5′ to 3′ direction.
Figure 2
Figure 2
Real-time PCR of miRNA precursor isoforms. The sequences of six miRNA precursor isoforms (let-7a-1, let-7a-2, let-7a-3, let-7f-1, let-7f-2 and let-7d) were cloned into plasmids. Real-time PCR was attempted on seven different reactions (in triplicate) containing each plasmid and primers specific to each isoform. Each reaction contained the TaqMan MGB probe for let-7d. Only the reaction containing the let-7d plasmid gave a detectable signal (A). Following the real-time PCR, a portion of each reaction was run on an agarose gel to demonstrate that PCR had occurred in each reaction (B). NTC, no template control. M, 100 bp DNA ladder.
Figure 3
Figure 3
Discrimination of let-7 miRNA precursor isoforms. Real-time PCR was attempted on miRNA precursor genes contained within plasmids using gene-specific primers and TaqMan MGB probes. The relative detection was calculated based upon the CT difference between the perfectly matched and mismatched targets.
Figure 4
Figure 4
U6 RNA expression in cancer cell lines. The expression of U6 RNA internal control in lung (orange), prostate (green), head and neck (yellow), breast (brown), colorectal (blue), hematopoietic (violet) and pancreatic (grey) cancer cell lines. Dashed line, mean U6 value.
Figure 5
Figure 5
Heatmap of miRNA precursor expression in 32 human cancer cell lines. (A) The names of the 32 cancer cell lines are listed on the top of the figure. The names of the miRNAs that were profiled in the cancer cell lines are listed to the right of the figure. The relative expression of each gene was determined by real-time PCR; data are presented as ΔCT. Unsupervised hierarchical clustering was performed using PCR primers to 201 miRNA precursors. Data were unfiltered prior to clustering. A median expression value equal to one was designated black; red increased expression; green, reduced expression; grey, undetectable expression. (B) Dendrogram of clustering analysis.
Figure 6
Figure 6
Validation of mature miRNA expression by northern blotting. The expression of three mature miRNAs and U6 RNA was validated in eight cancer cell lines by northern blotting. (A) Northern blot of mature miR-100, miR-21, miR-205 and U6 RNA in the eight cancer cell lines listed. The identical blot was stripped and re-probed for each of the RNAs. (B) Densitometric analysis of the mature miRNA (northern blot) is compared with the miRNA precursor expression as determined by the real-time PCR assay.
Figure 7
Figure 7
Reduced expression of tissue-specific miRNAs in continuous cell lines compared with tissues. The expression of miR-122a and miR-1-1/miR-133 were reported previously to be specific to liver and muscle tissue, respectively. (A) Northern blotting to miR-122a was performed on RNA isolated from specimens of hepatocellular carcinoma from three different patients (T), normal liver tissue (N) and in the liver cancer cell lines SK-Hep1, PLC/PRF5, SNU387 and SNU449, and lung cancer cell line H719. The blots were stripped and re-probed for U6 RNA. (B) Total RNA from the rhabdomyosarcoma cell lines RD2, SMS-CTR, CW9019 and RH30 and normal skeletal muscle tissue (N) was probed for miR-1-1, miR-133 and U6 RNA.
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References

    1. Ambros V. The functions of animal microRNAs. Nature. 2004;431:350–355. - PubMed
    1. Bartel D.P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–297. - PubMed
    1. Lee Y., Ahn C., Han J., Choi H., Kim J., Yim J., Lee J., Provost P., Radmark O., Kim S., et al. The nuclear RNase III Drosha initiates microRNA processing. Nature. 2003;425:415–419. - PubMed
    1. Griffiths-Jones S. The microRNA Registry. Nucleic Acids Res. 2004;32:D109–D111. - PMC - PubMed
    1. Babak T., Zhang W., Morris Q., Blencowe B.J., Hughes T.R. Probing microRNAs with microarrays: tissue specificity and functional inference. RNA. 2004;10:1813–1819. - PMC - PubMed

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