Nanodrop 2000c spectrometer

BL21(DE3) and B-95.ΔA were transformed with pET-Fab and pET-Fab(Am121). The transformed cells were cultured in 5 ml of the auto-induction medium, with vigorous shaking at 25°C for 22 hours. The harvested cells were lysed in a BugBuster Master Mix solution (Novagen) using 500 μl solution per 100 mg cells. The lysate (400 μl) was mixed with Protein G Mag Sepharose Xtra magnetic beads (100 μl) (GE Healthcare), as described previously45 (link). Fab fragments were eluted in an acidic condition, and the eluate (100 μl) was then neutralized by the buffer included in the Ab buffer kit (20 μl) (GE Healthcare). The yields of Fab fragments were determined based on the absorption at 280 nm, using a NanoDrop 2000c spectrometer (Thermo Scientific). The purified Fab was fractionated on a NuPAGE gel and detected using the SimplyBlue SafeStain (Life Technologies). The Rhodamine-triarylphosphine (RTP) conjugate and the FITC-triarylphosphine (FTP) conjugate were commercially synthesized by Shinsei Chemical Co. Ltd. (Osaka, Japan). The RTP and FTP were dissolved in dimethyl sulfoxide at the concentrations of 4 mg/ml and 5 mM, respectively. The solution containing the purified Fab was mixed with one-twentieth volume of RTP or FTP, and then incubated at 37°C for 30 minutes. The labeled Fab fragments were fractionated on a NuPAGE gel and then visualized using an LAS4010 spectrometer (GE Healthcare).

The PCR products were purified using the Wizard SV Gel and PCR clean-up system kit (Promega, Germany) and its concentration determined using the NanoDrop 2000c spectrometer (Thermo Scientific, USA). The concentration of each purified product was adjusted and prepared according to the instruction recommended by the sequencing company and submitted for sequencing along with sequencing primers (forward and reverse) to LGC Genomics (Berlin, Germany).

Gene Jet RNA Purification kit (Thermo Scientific) was used to isolate total ribonucleic acid (RNA) according to the manufacturer’s instructions. RNA was quantified on a Thermo Scientific Nanodrop 2000c Spectrometer and considered pure if the ratio of the absorbance at 260 nm/280 nm was ≥ 2. Then, samples were stored at −20 °C until they were analyzed for RT-PCR. The RNA was prepared as a template for complementary deoxyribonucleic acid (cDNA) synthesis using the iScript cDNA Synthesis kit (Bio-Rad). Quantitative RT-PCR analysis was performed using 10.4 ng of cDNA per reaction and SYBER^® Green PCR Supermix (Bio-Rad). Gene expression was normalized to the housekeeping gene GAPDH and the control group (2^−ΔΔC). Gene expression values were calculated using the mean cycle threshold (CT) values of the samples. All primers (Table S2) were synthesized by Integrated DNA Technologies (Coralville, IA, USA).

Total RNA was extracted from NIH3T3 cells by Eastep^® Super Total RNA Extraction Kit (Promega, China) according to the manufacturer’s instructions. The concentration and purity of extracted RNA samples were detected by Nanodrop 2000C spectrometer (Thermo Scientific, USA). Then, total RNA was reversely transcribed to cDNA with a reverse transcription Kit (Promega, China). QPCR was then performed on a QuantstudioTM Dx Real‐Time PCR System (Thermo Fisher Scientific, USA) by using SYBR^® Green Premix Pro Taq HS qPCR Kit (Agbio, China). The program was run with the following settings: preheated at 95°C for 30 s, denatured at 95°C for 5 s, annealed at 60°C for 30 s. The total of 40 cycles were repeated. The Ct values of the target genes were corrected by using the internal reference Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of the same sample. The synthesized primers are shown in Table 2.

This study comprised 41 cervical tissue samples. We obtained 26 formalin-fixed paraffin-embedded (FFPE) and 15 frozen cervical tissues samples from the Korea Gynecologic Cancer Bank, Yonsei University College of Medicine, Seoul, Republic of Korea. We divided the collected samples into three control groups (HPV-negative normal tissues [NN], HPV-negative cancer tissue [NC], and HPV16-positive normal tissue [PN]) and one experimental group (HPV16-positive cancer tissue [PC]). Detailed information about these samples is reported in Table 1. All cancer samples were squamous cell carcinomas, which comprise about 80% of all cervical cancers. HPV infection was confirmed using the Abbott RealTime High-Risk HPV PCR assay kit (Abbott Molecular, Abbott Park, IL, USA).
Total RNA was extracted from the frozen tissues using the Labozol reagent (CosmoGenetech, Seoul, South Korea) and from the FFPE tissues using the miRNeasy FFPE kit (Qiagen, Valencia, CA, USA). RNA was quantitated using a Nanodrop2000c spectrometer (Thermo Scientific, Wilmington, DE, USA) and DS-11 (DeNovix, Wilmington, DE, USA) spectrophotometers, and the quantitation and quality of the isolated samples were confirmed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).