Lightcycler instrument

Total RNA was isolated from LncRNA RAB5IF depleted or intact HepG2 and Hep3B cells by using RNeasy mini kit (Qiagen) and reverse-transcribed using M-MLV reverse transcriptase (Promega, Madison, WI). Quantitative reverse transcription PCR (RT-qPCR) was conducted with the LightCycler TM instrument (Roche Applied Sciences, Indianapolis, IN, USA) using the following primers, LncRNA RAB5IF- forward: 5′-AGTCTCCGTCTGGAGTAAGGTG−3′; reverse- 5′-CCTGCTATTCCCAAGAACCCTC–3′ (Bioneer, Daejeon, Korea), LGR5 forward: 5′- CGTTGCAACACTGTCATGGC-3′; reverse- 5- AGGTCAGGTGAAGCGCTCG−3′ (Bioneer, Daejeon, Korea), hGAPDH-forward 5′-CCA CTC CTC CAC CTT TGA CA-3′; reverse-5′-ACC CTG TTG CTG TAG CCA −3′ (Bioneer, Daejeon, Korea).

Total RNAs were isolated using the RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions and reverse transcribed using M-MLV reverse transcriptase (Promega, Madison, WI, USA). The cDNAs were amplified by PCR using the synthesized specific primers (Bioneer, Daejeon, Korea) (Table 1).
RT-qPCR was operated with the lightcycler TM instrument (Roche Applied Sciences, Indianapolis, IN, USA) according to the manufacturer’s protocol. PCR started at 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 57 °C for 15 s, and 72 °C for 20 s. The mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize the expression of genes of interest. Each sample was tested in triplicate, and relative gene expression data were analyzed by means of the 2^−ΔCT method.

Total RNA from prostate cells was isolated using the QIAzol (Invitrogen, Carlsbad, CA, USA) and one microgram of total RNA was used to make cDNA by Superscript reverse transcriptase and amplified by Platinum Taq polymerase with Superscript One Step RT-PCR kit (Invitrogen, Carsbad, CA,USA). Primers sequences used were synthesized by Bioneer (Daejeon, Korea) with the following sequences: hDR5-forwad-5'-GTC TGC TCT GAT CAC CCA AC-3', reverse-5'-CTG CAA ACT GTG ACT CCT ATG-3', hGAPDH -forward - 5'-TCA CCA TCT TCC AGG AGC GA-3'; reverse -5'-CAC AAT GCC GAA GTG GTG GT-3'. For PCR amplification, following conditions was used; an initial step at 50 °C for 30 min, 94 °C for 2 min, followed by 30 cycles at 94°C for 15 s, 55°C for 30 s and 72°C for 1 min, and a final step at 72°C for 10 min. The amplified products were separated on 2% agarose gel. For RT-qPCR, RT-qPCR was performed with the LightCycler TM instrument (Roche Applied Sciences, Indianapolis, IN) with following primers, hDR5- forward: 5'- GAC TCT GAG ACA GTG CTT CGA TGA -3'; reverse- 5'-CCA TGA GGC CCA ACT TCC T-3', hGAPDH-forward5'-CCA CTC CTC CAC CTT TGA CA-3';reverse-5'-ACC CTG TTG CTG TAG CCA -3'.

The total RNA of cells was isolated from HepG2 cells using QIAZOL lysis reagent (QIAZEN, Venlo, Netherlands). After synthesis process of cDNA by using M-MLV reverse transcriptase (Promega, WI, USA), the mRNA levels were measured by qRT-PCR with the light cycler TM instrument (Roche Applied Sciences, IN, USA) according to manufacturer’s protocol. The mRNA level of GAPDH was used to normalize the expression of genes of interest. Primers of MID1IP1, SREBP-1c and FAS were purchased from Bioneer. The sequences of these primers used are as follows (Table 1):
Each sample was tested in triplicates, and relative gene expression data were analyzed by means of the 2^−∆CT method.

Total RNA was isolated by using RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions and reverse transcribed using M-MLV reverse transcriptase (Promega, Madison, WI). The cDNA was amplified by PCR using the synthesized specific primers as follows (Bioneer, Daejeon, Korea): c-Myc forward 5′-CCACCAGCAGCGACTCTGA-3′ and reverse 5′-GCAGAAGGTGATCCAGACTC-3′; E2F1 forward 5′-TCCAAGAACCACATCCAGTG-3′ and reverse 5′-CTGGGTCAACCCCTCAAG-3′; CCND1 forward 5′- GAAGATCGTCGCCACCTG-3′ and reverse 5′-GACCTCCTCCTCGCACTTCT-3′; CDC25A forward 5′- ATCTCTTCACACAGAGGCAGAA-3′ and reverse 5′- CCCTGGTTCACTGCTATCTCTT-3′; NCL forward 5′-GTGGTGGACGGTGTTCACTT-3′ and reverse 5′-GCCACGGCCAGCACATCAT-3′; GAPDH forward 5′-CTGCACCACCAACTGCTTAG-3′ and reverse 5′-AGGTCCACCACTGACACGTT-3′.
RT-qPCR was operated with the light cycler TM instrument (Roche Applied Sciences, Indianapolis, IN) according to the manufacturer’s protocol. The mRNA level of GAPDH was used to normalize the expression of genes of interest.

The acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and beta-secretase (BACE1) genes expression level was analyzed by two-step quantitative real-time PCR (qRT-PCR), in a volume of 10 μL reaction mixture, using relative quantification methodology with a LightCycler TM Instrument (Roche, Germany) and a LightCycler Fast Start DNA Master SYBR Green I kit (Roche Applied Science) according to the instructions of the manufacturer. All primers sequences were designed and custom-designed using the Oligo 6.0 software (National Biosciences) and were verified by assessment of a single PCR product on agarose gel and by a single temperature dissociation peak (melting curve analysis) of each cDNA amplification product. An GAPDH gene was used as a housekeeping gene (endogenous internal standard) for normalization of qPCR. For each quantified gene, standard curves were prepared from dilution of cDNA and generated from a minimum of four data points. All quantitative PCR were repeated twice. The data were evaluated using LightCycler Run 4.5 software (Roche Applied Science). Each PCR run included a nontemplate control to detect potential contamination of reagents.

Based on Lee et al.’s paper [27 (link)], with the total RNAs isolated from CK-treated DU145 cells by using RNeasy mini kit (Qiagen), quantitative reverse transcription PCR (RT-qPCR) was carried out under the LightCycler TM instrument (Roche Applied Sciences, Indianapolis, IN, USA) by using the following primers, IL-6- forward: 5′-CCACCGGGAACGAAAGAGAA−3‘; reverse-: 5′-GAGAAGGCAACTGGACCGAA–3′ (Bioneer, Daejeon, Korea); IL-10 forward: 5′-TCTCCGAGATGCCTTCAGCAGA-3′; reverse-: 5′-TCAGACAAGGCTTGGCAACCCA−3′ (Bioneer, Daejeon, Korea); hGAPDH-forward: 50 -CCA CTC CTC CAC CTT TGA CA-30; reverse-: 50 -ACC CTG TTG CTG TAG CCA −3 0 (Bioneer, Daejeon, Korea).