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

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 were purified using the QIAquick

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 were used for negative control.

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

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

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

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

## **4. Study designs**

280 Bioinformatics

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.

We classified all the candidate miRNAs using the PhastCon database at the University of California at Santa Cruz.**(10, 11)** This database has scores for each nucleotide in the human genome relative to its degree of conservation when compared to nucleotides in the armadillo, bush baby, cat, chicken, chimpanzee, cow, dog, elephant, frog, fugu, guinea pig, hedgehog, horse, lizard, medaka, mouse, opossum, platypus, rabbit, rat, rhesus monkey, shrew, stickleback, tenrec, tetraodon, tree shrew, and zebrafish. The algorithm is based on a phylogenetic hidden Markov model that uses best-in-genome pairwise alignment for each species (based on BLASTZ), followed by multiple alignment of the twenty eight genomes. A hairpin was defined as conserved if the average PhastCon conservation score over the 28

Real-time PCR was performed on an ABI7300 (Applied Biosystems, Foster City, CA, USA) using various mirVana qRTPCR primer sets (Ambion, Austin, TX, USA) and a SYBR ExScript RT-PCR kit (Takara Bio), or with TaqMan miRNAs assays (Applied Biosystems), a High capacity cDNA archive kit (Applied Biosystems), and Absolute QPCR ROX mix (Abgene, Rochester, NY, USA), according to the manufacturers' instructions. As an

After bioinformatic analysis of the sequence data, we further validated novel miRNAs by using a combination of Ago2-immunoprecipitation **(13)** followed by PCR-based miRNA detection (**14**). Briefly, 50 ml Dynabeads protein G slurry (Invitrogen) was immobilized with 20 mg human P4 anti-mouse Ago2 monoclonal antibody (clone 2D4, Wako Pure Chemical Industries, Osaka, Japan). One hundred fifty micrograms of human tissue P4 were homogenized in 1.5 ml of a cell lysis solution (provided in miRNAs isolation kit, Wako) using a Polytron PT1200C homogenizer (Kinematica AG, Lucerne, Switzerland) for 10 s at 4 8C, and then 1.5 ml of the cell lysis solution was added into the homogenized solution. Following incubation for 15 min on ice, lysate was centrifuged at 20 000 g for 20 min at 4 8C and filtered through a 0.8 mmSupor Acrodisc syringe filter (Pall Corporation, Ann Arbor, MI, USA). One milliliter of the filtered lysate was incubated with 25 ml of the anti-Ago2- Dynabead protein G for incubation for 60 min at 4 8C. After immunoprecipitation, Ago2-

*2.2.3. Determination of hairpin conservation of Novel MicroRNAs* 

species for any 15-nt sequence in the hairpin stem was at least 0.8 .**(12)**

**3.2. Ago2-immunoprecipitation and PCR analysis of novel miRNAs** 

**3. Other techniques for MicroRNAome** 

**3.1. Real-time PCR analysis of known miRNAs** 

endogenous control, 5SrRNA or U6 snRNA was used.

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.
