**2.1. MicroRNA cloning**

The method for microRNA cloning and sequencing that we moderated from the original ones are shown in Fig1. We cloned small RNA by a modification of the published miRNA cloning protocol of Lagos-Quintana et al. **(6)**. In brief, total RNA samples were extracted using ISOGEN (Nippon Gene, Tokyo, Japan), separated in a denaturing polyacrylamide gel, and the 18–24 nt fraction was recovered. Next, 5′- and 3′-adapters were ligated to the RNAs Ligation of small RNAs with DNA\_RNA chimera linkers at both termini [3' linker

© 2012 Mizuguchi et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Mizuguchi et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

oligonucleotide (5'/5 Phos/rCrUrGrUAGGCACCATCAATdi-deoxyC-3 ') and the 5' linker oligonucleotide (5'-ATCGTrArGrGrCrArCrCrUrGrArArA-3')] and RT-PCR was carried out.

Novel microRNA Cloning Using Bioinformatics 279

*2.2.1. Comparing the cloned sequences with those of known RNAs* 

to further analysis. The result is shown in table1.

**Table 1.** Annotation of the sequenced small RNAs

*2.2.2. Secondary structure analysis of Novel microRNAs* 

The vector sequence, the 5'and 3' linkers, and their coupled sequences (CTGTAGGCACCTGAAA) were removed. Those extracted sequences composed of 16–30 nt were defined as valid small RNAs and were subjected to followings. The small RNA sequences were analyzed for homology with known RNAs, including miRNA, piwiinteracting (pi) RNA, rRNA, tRNA, small nuclear (sn) RNA, small nucleolar (sno) RNA, and mRNA, and human genomic DNA sequences. The databases used were: miRNA (matureand pre-), Sanger Database.; piRNA, the NCBI Entrez Nucleotide database; rRNA, the European ribosomal RNA database; tRNA, the Genomic tRNA database; sn/snoRNA, RNAdb and NONCODE; mRNA, NCBI Reference Sequence; and human genomic sequences, the UCSC Genome Bioinformatics Site. In our searches, we defined the cloned sequencing results that had higher than 90% homology as valid if they met our criteria for sequence error, erroneous PCR amplification, and 3′- and 5′-end variations. Clones with 100% homology with human genomic DNA but did not match known RNAs when compared to the above databases were termed novel miRNA candidates and were subjected

Reads (%) HCC ANL Sum Total 314,359 268,708 583,067 miRNA 256,64 (81.6) 208,038 (77.4) 464,687 (79.7) piRNA 2,983 (0.9) 1,440 (0.5) 4,423 (0.8) rRNA 5,474 (1.7) 10,161 (3.8) 15,635 (2.7) tRNA 1,703 (0.5) 621 (0.2) 2,324 (0.4)

snRNA 700 (0.2) 343 (0.1) 1,043 (0.2)

snoRNA 654 (0.2) 747 (0.3) 1,401 (0.2) mRNA 6,053 (1.9) 7,279 (2.7) 13,332 (2.3) Genome 2,799 (0.9) 3,149 (1.2) 5,948 (1.0) Others 15,588 (5.0) 34,686 (12.9) 71,174 (12.2)

The two-dimensional precursor miRNA (pre-miRNA) configurations of our novel miRNA candidates were predicted according to the method described previously **(8)** with some modifications. Briefly, 196-nt of genomic sequence was added to the candidate sequences (88-nt at each end). Each candidate sequence was divided into 110-nt windows and subjected to two-dimensional analysis along its entire length, using the RNAfold software (Vienna RNA Secondary Structure Package **(9)**). The configurations that had the lowest free energy and that had a high conservation (described below) and met the following criteria were termed novel miRNAs: (*a*) contained a stem-loop configuration; (*b*) cloned mature

**Figure 1.** Overview of the miRNA cloning. See paper body for details.

Amplification of the cDNA fragments was obtained by two consecutive rounds of PCR. Specific restriction enzyme digestion of the adaptors allowed for concatemerization of the cDNA into larger fragments. These fragments were then cloned into a vector to create a cDNA library. Concatemerization increases the length of informative sequences obtainable from each clone. we concatenated more than 20 cDNAs into a single fragment using a BanI restriction enzyme (New England Biolabs, Ipswich, MA, USA), a DNA ligation kit ver. 2.1 (Takara Bio, Shiga, Japan), and a Geneclean III kit (Qbiogene, Irvine, CA, USA) prior to TA cloning. The concatenated products were then inserted into plasmids and sequenced (Fig1).

The sequences were compared to human DNA to determine the genomic origin of the small RNA. It was important to avoid contamination from other samples and molecular-weight makers during electrophoresis. Such contaminants considerably diminished the accuracy and efficiency of miRNA cloning. We avoided contamination by performing the cloning procedure separately for each sample, by using a special gel with a small plastic rod that divided the sample and marker lanes, and by using separate vats for each gel for ethidium bromide staining. We made small RNA libraries by excising a portion of a polyacrylamide gel containing species 18–24 nt in length to avoid contaminating our purified RNAs with piRNAs **(7)**.

#### **2.2. Bioinformatics analysis of the sequence data**

We performed a homology search for all cloned small RNAs and a secondary structural analysis for all novel miRNA candidates.

#### *2.2.1. Comparing the cloned sequences with those of known RNAs*

278 Bioinformatics

piRNAs **(7)**.

oligonucleotide (5'/5 Phos/rCrUrGrUAGGCACCATCAATdi-deoxyC-3 ') and the 5' linker oligonucleotide (5'-ATCGTrArGrGrCrArCrCrUrGrArArA-3')] and RT-PCR was carried out.

Amplification of the cDNA fragments was obtained by two consecutive rounds of PCR. Specific restriction enzyme digestion of the adaptors allowed for concatemerization of the cDNA into larger fragments. These fragments were then cloned into a vector to create a cDNA library. Concatemerization increases the length of informative sequences obtainable from each clone. we concatenated more than 20 cDNAs into a single fragment using a BanI restriction enzyme (New England Biolabs, Ipswich, MA, USA), a DNA ligation kit ver. 2.1 (Takara Bio, Shiga, Japan), and a Geneclean III kit (Qbiogene, Irvine, CA, USA) prior to TA cloning. The concatenated products were then inserted into plasmids and sequenced (Fig1). The sequences were compared to human DNA to determine the genomic origin of the small RNA. It was important to avoid contamination from other samples and molecular-weight makers during electrophoresis. Such contaminants considerably diminished the accuracy and efficiency of miRNA cloning. We avoided contamination by performing the cloning procedure separately for each sample, by using a special gel with a small plastic rod that divided the sample and marker lanes, and by using separate vats for each gel for ethidium bromide staining. We made small RNA libraries by excising a portion of a polyacrylamide gel containing species 18–24 nt in length to avoid contaminating our purified RNAs with

We performed a homology search for all cloned small RNAs and a secondary structural

**Figure 1.** Overview of the miRNA cloning. See paper body for details.

**2.2. Bioinformatics analysis of the sequence data** 

analysis for all novel miRNA candidates.

The vector sequence, the 5'and 3' linkers, and their coupled sequences (CTGTAGGCACCTGAAA) were removed. Those extracted sequences composed of 16–30 nt were defined as valid small RNAs and were subjected to followings. The small RNA sequences were analyzed for homology with known RNAs, including miRNA, piwiinteracting (pi) RNA, rRNA, tRNA, small nuclear (sn) RNA, small nucleolar (sno) RNA, and mRNA, and human genomic DNA sequences. The databases used were: miRNA (matureand pre-), Sanger Database.; piRNA, the NCBI Entrez Nucleotide database; rRNA, the European ribosomal RNA database; tRNA, the Genomic tRNA database; sn/snoRNA, RNAdb and NONCODE; mRNA, NCBI Reference Sequence; and human genomic sequences, the UCSC Genome Bioinformatics Site. In our searches, we defined the cloned sequencing results that had higher than 90% homology as valid if they met our criteria for sequence error, erroneous PCR amplification, and 3′- and 5′-end variations. Clones with 100% homology with human genomic DNA but did not match known RNAs when compared to the above databases were termed novel miRNA candidates and were subjected to further analysis. The result is shown in table1.


**Table 1.** Annotation of the sequenced small RNAs

#### *2.2.2. Secondary structure analysis of Novel microRNAs*

The two-dimensional precursor miRNA (pre-miRNA) configurations of our novel miRNA candidates were predicted according to the method described previously **(8)** with some modifications. Briefly, 196-nt of genomic sequence was added to the candidate sequences (88-nt at each end). Each candidate sequence was divided into 110-nt windows and subjected to two-dimensional analysis along its entire length, using the RNAfold software (Vienna RNA Secondary Structure Package **(9)**). The configurations that had the lowest free energy and that had a high conservation (described below) and met the following criteria were termed novel miRNAs: (*a*) contained a stem-loop configuration; (*b*) cloned mature

miRNA sequence portion consisted of more than 16-nt in its double-stranded region; (*c*) the loop contained fewer than 20-nt; (*d*) the internal loop contained fewer than 10-nt; and (*e*) the bulge contained fewer than 5-nt. Furthermore, novel sequences with overlapping positions in the genome were grouped together. Novel antisense miRNAs are defined with above criteria (a)-(e) but without conservation score if they are coded in same chromosomal region. Novel microRNA Cloning Using Bioinformatics 281

associated RNAs were isolated from the immunoprecipitate according to the manufacture's protocol (Wako). We confirmed that the immunoprecipitate contained human P5 Ago2 protein of w100 kDa in size by western blot (data not shown). Non-immune human IgG (Sigma) was used as a control for Ago2-immunoprecipitation. Preparation of the cDNA library using the Ago2-associated RNAs and semi-quantitative PCR analysis of the abovementioned novel miRNA candidates were performed, as reported previously **(14)**. A small RNA-specific primer and a universal reverse primer RTQ-UNIr **(14)**, were used for amplification of each of the small RNAs. The PCR products were analyzed on a 12% polyacrylamide gel. The primers for the human GAPDH were used for negative control.

After bioinformatic analysis of the sequence data, we further validated novel miRNAs by PCRbased miRNA detection **(14).** Briefly, small RNAs were isolated using the mirVana™ miRNA isolation kit (Ambion). Small RNA samples were polyadenylated with Poly(A) Tailing Kit (Ambion) and were purified with Acid-Phenol:Chloroform and with filter cartridge provided in the mirVana Probe & Marker Kit (Ambion). To generate a small RNA cDNA library, tailed RNA were reverse transcripted using RTQ primer**(14)** and the samples

spin PCR purification kit (QIAGEN). A small RNA-specific primer and a universal reverse primer RTQ-UNIr **(14)**, were used for amplification of each of the small RNAs. The PCR products were analyzed on a 12% polyacrylamide gel. The primers for the human GAPDH

Total miRNA (350 ng) was reverse-transcribed using Megaplex RT Primers (Applied Biosystems). The resulting cDNAs were pre-amplified using Megaplex PreAmp Primers (Applied Biosystems) and the pre-amplified products applied to a TaqMan Human

Cultured cells were transfected with precursor hsa-miR-200c and hsa-miR-141 (ID: PM11714; PM10860); Anti-miR™ 200c and 141 inhibitors (ID: MH11714; MH10860) (Ambion, Austin, TX) for 8 hours in serum free medium. Serum supplemented medium was

Sequencing using 454 sequencing and conventional cloning from 22 pair of HCC and adjacent normal liver (ANL) and 3 HCC cell lines identified reliable reads of more than 300000 miRNAs from HCC and more than 270000 from ANL for registered human miRNAs.

added and gene and protein expression measured at the indicated time points.

**3.3. PCR analysis of novel miRNAs (Alternative method of 3.2)** 

**3.4. Real-time PCR-based miRNA expression profiling** 

**3.5. siRNA, Pre-miR and anti-miR transfections** 

were purified using the QIAquick

were used for negative control.

**4. Study designs** 

MicroRNA Array Panel (A and B, v2.0).
