**5. Detected novel microRNAs using cloning**

Eleven novel opposite miRNAs (defined as miRNAs cloned from the other arm of precursors from which known miRNAs have been cloned) were identified from the annotated miRNAs (Figure 2A).Based on above criteria, a scan of all the novel miRNA candidates identified 245 novel precursors, representing putative 210 novel mature miRNAs (Figure 2B).

Novel microRNA Cloning Using Bioinformatics 283

Precursor

score

dG (kcal/mol)

Start End PhastCon

Accession

the precursor.

Number Most frequent sequence Strand Chr.

**Table 2.** Summary of the novel miRNAs

AB372573 TGCAATGGTTGTCCTGAGA + 1 30294032 30294114 0.00 -28.6 AB372574 GTGGGGGAGAGGCTGTCA + 1 150066781 150066870 0.00 -50.7 AB372575 TTGCACAACTCTCACATTC + 1 152498870 152498979 0.39 -38.2 AB372576 CCCCTCACTTCTCCGTCATATC + 1 153367241 153367332 0.05 -28.8 AB372577 CCCCAGATTCCACACC + 1 154326362 154326427 0.85 -28.9

Summary of predicted novel miRNAs. The predicted novel miRNAs with their PhastCon scores are listed. And the most frequent cloned sequences, genome locations, clone counts, and conservation

**Table 3.** Samples of the Secondary structures of predicted novel miRNAs. The predicted novel miRNAs with their particular ID numbers are listed. The mature miRNAs are depicted in blue in the drawing of

scores calculated with PhastCon are listed. The stem-loop is a 110-nt sequence derived by

computational prediction. The precursor structures are listed in Table 3.

**A:** Chromosomal locations of our cloned mature miRNAs which correspond to previously identified pre-miRNAs. The bar graphs indicate the location of mature miRNAs (both our cloned miRNAs and previously identified miRNAs) based on known pre-miRNA sequences. Many of our cloned sequences were found in these regions. Arrowheads indicate novel "opposite" miRNAs cloned in our experiments. These sequences represent mature miRNAs which originate on the opposite DNA strand of the known precursor miRNA.

**B:** The 222 novel mature miRNAs identified in our study were checked for species conservation using the PhastCon database and their corresponding hairpin conservation scores charted in this figure.

**Figure 2.** Qualitative analysis of miRNAs in the human liver.


#### **Table 2.** Summary of the novel miRNAs

282 Bioinformatics

(Figure 2B).

**5. Detected novel microRNAs using cloning** 

Eleven novel opposite miRNAs (defined as miRNAs cloned from the other arm of precursors from which known miRNAs have been cloned) were identified from the annotated miRNAs (Figure 2A).Based on above criteria, a scan of all the novel miRNA candidates identified 245 novel precursors, representing putative 210 novel mature miRNAs

**A:** Chromosomal locations of our cloned mature miRNAs which correspond to previously identified pre-miRNAs. The bar graphs indicate the location of mature miRNAs (both our cloned miRNAs and previously identified miRNAs) based on known pre-miRNA sequences. Many of our cloned sequences were found in these regions. Arrowheads indicate novel "opposite" miRNAs cloned in our experiments. These sequences represent mature miRNAs which

**B:** The 222 novel mature miRNAs identified in our study were checked for species conservation using the PhastCon

originate on the opposite DNA strand of the known precursor miRNA.

**Figure 2.** Qualitative analysis of miRNAs in the human liver.

database and their corresponding hairpin conservation scores charted in this figure.

Summary of predicted novel miRNAs. The predicted novel miRNAs with their PhastCon scores are listed. And the most frequent cloned sequences, genome locations, clone counts, and conservation scores calculated with PhastCon are listed. The stem-loop is a 110-nt sequence derived by computational prediction. The precursor structures are listed in Table 3.

**Table 3.** Samples of the Secondary structures of predicted novel miRNAs. The predicted novel miRNAs with their particular ID numbers are listed. The mature miRNAs are depicted in blue in the drawing of the precursor.

We cloned 210 novel microRNA candidates. Samples of the novel microRNAs that were detected from our study and its bioinformatics data are tabulated in Table 2 and 3. And those novel miRNAs have been deposited with DDBJ under consecutive accession codes from AB372573 to AB372814.

Novel microRNA Cloning Using Bioinformatics 285

Takuya Mishima and Toshihiro Takizawa

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