Sybr green 1 qpcr kit

Total RNA was isolated from the cells using an Easy-BLUE RNA extraction kit (iNtRON Biotech, 17061). Standard RT was performed using an amfiRivert cDNA Synthesis kit (GenDEPOT, R5101) according to the manufacturer’s instructions. qRT-PCR measurements were performed using the ABI Prism 7000 Sequence Detection System (Applied Biosystems) and a SYBR Green I qPCR kit (Takara, RR420) according to the manufacturers’ instructions. The cDNA was amplified with the following primers: 5′-GAAGAAGCTGTGTGGCACTG-3′ and 5′-GTTCGGTCAGAAATGGCTTC-3′ for mouse cathepsin B, 5′-AGGTGAAGGAGCTGCAGAAG-3′ and 5′-ATTCCCATGAAGCCACTCAG-3′ for mouse cathepsin D, and 5′-AACGGGAAGCCCATCACC-3′ and 5′-CAGCCTTGGCAGCACCAG-3′ for mouse GAPDH and 5′-GATCGCACAGGTCTTTAAGAA -3′ and 5′-CATCGACGTTGGGTAGAC AC-3′ for rat cathepsin B, 5′-ACACTGTGTCGGTTCCATGT -3′ and 5′-TGCGATGAATACGACTCCAG -3′ for rat cathepsin D, and 5′-CAA AGACCGGAACAACCACT -3′ and 5′-CCTTCGGATGTAGTGTCCGT-3′ for rat cathepsin L and 5′-GTTACCAGGGCTGCCTTCTC -3′ and 5′-GGGTTTCCCGTTGATGACC-3′ for rat GAPDH. With normalization to GAPDH and GUSB, the relative gene expression was analyzed using the comparative threshold cycle (CT) method (Applied Biosystems). The expression of the genes of interest was expressed as fold change over control.

The total RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction, and the complementary DNA (cDNA) was synthesized using a RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas, Hanover, NH, USA). qRT-PCR was processed using a SYBR Green I qPCR Kit (TaKaRa, Shiga, Japan), via the CFX Connect™ Real-Time System (Bio-Rad). The gene-specific primers used in this study were as follows: forward 5′-TCC CAC GTT TTC ACA TTC GG-3′ and reverse 5′-CCC GTT ACC ATA TAG GAT AGC C-3′ for mouse Alpl, forward 5′-TAA AGT GAC AGT GGA CGG TCC C-3′ and reverse 5′-AAT GCG CCC TAA ATC ACT GAG G-3′ for mouse Runx2, forward 5′-AAT ACC TCC CTC TCG ATC CTA CA-3′ and reverse 5′-TGG TTC TTG ACT GGA GTA ACG TA-3′ for mouse Ctsk, forward 5′-TGG TAT GTG CTG GCT GGA AAC-3′ and reverse 5′-AGT TGC CAC ACA GCA TCA CTG-3′ for mouse Acp5, forward 5′-AGG TCG GTG TGA ACG GAT TTG-3′ and reverse 5′-TGT AGA CCA TGT AGT TGA GGT CA-3′ for mouse Gapdh, forward 5′-GAG GAG TCC TGT TGA TGT TGC CAG-3′ and reverse 5′-GGC TGG CCT ATA GGC TCA TAG TGC-3′ for mouse Hprt. All gene expression levels were normalized using mouse Gapdh (osteoblast) and mouse Hprt (osteoclast), and relative expression levels were calculated using the 2^−ΔΔCt method (ΔΔCt = ΔCt_Treatment − ΔCt − _Induction).

Total RNA was harvested using QIAzol reagent (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed using 100 ng of cDNA and the SYBR Green I qPCR kit (TaKaRa, Shiga, Japan). Fluorescence-based amplification was analyzed using a CFX Connect™ Real-Time System (Bio-Rad). The primers used in this study are listed in Supplementary Table S1. The expression levels of each gene were normalized to mouse Gapdh (for osteoblasts) and Hprt (for osteoclasts), and the relative quantification of the gene expression was calculated using the ΔΔCt method (ΔCt_Treatment − ΔCt_Induction). The fold-change is expressed as 2^−ΔΔCt.

Total RNAs from mouse pre-osteoblast MC3T3-E1 cells, mouse monocytes, and human samples were isolated using TRIzol reagent (Invitrogen; Carlsbad, CA, USA) and treated with RNase-free DNase I (Invitrogen). One microgram of total RNA was subsequently reverse-transcribed using the RevertAid™ Minus First Strand cDNA Synthesis Kit (Fermentas Inc.; Hanover, MD, USA) with both the oligo (dT) 15-18 primer and the random hexamer primer. The qRT-PCR was performed using the Qiagen Rotor-Q (Qiagen; Hilden, Germany) or CFX Connect™ Real-Time PCR Detection System (Bio-Rad Laboratories). PCR amplification (40 cycles) was performed with reaction mixtures of a total volume of 10 µl containing cDNA (100 ng) using the SYBR Green I qPCR kit (Takara Bio Inc.) according to the manufacturer’s instructions. Gene-specific primers (COSMOGENETECH, Seoul, Korea) used for qRT-PCR are shown in Supplementary Table 8.
Relative quantification of gene expression was performed using the cycle threshold (Ct) method as described by the manufacturer’s manual (Qiagen or Bio-Rad) and normalized to mouse Gusb or human GUSB expression. Relative gene expression of target genes was calculated as 2^{-delta (Δ) Ct} (ΔCt=Ct _{target gene} − Ct _{control gene}) and fold change was calculated as 2^-ΔΔCt (ΔΔCt = ΔCt _{target sample} − ΔCt _{reference sample}). The quantification value was expressed as the fold change relative to the control.

Total RNA was isolated from the cultured neurons with TRIzol according to the instructions. RNA was reversely transcripted to cDNA. The SYBR Green I qPCR kit (Takara, Japan) was used for quantitative PCR following the manufacturer’s protocol. qPCR was detected in triplicate. The gene expression of each sample was normalized to the expression of GAPDH. The qPCR condition was incubated at 95°C for 10 min before 45 cycles of 95°C for 10 s, 63°C for 5 s and 72°C for 15 s. Melting curve was analyzed at 65°C, with the temperature increasing at a rate of 1°C every 10 s to 95°C. The primers used for the research were as follows: Netrin-1, 5′-CCGTGGTGACCAGAGTTTGT-3′ and 5′-ATCACCAGGCTGCTCTTGTC-3′; UNC5B, 5′-CGACCCTAAAAGCCGCCCC-3′ and 5′-GGGATCTTGTCGGCAGAGTCC-3′; DCC, 5′-ACATCCGACGTTCGGCTTT-3′ and 5′-TGATTTTCCCATTGGCTTCC-3′; GAPDH, 5′-CCCTTCATTGACCTCAACTA-3′ and 5′-CCAAAGTTGTCATGGATGAC-3′. GAPDH was used as the reference gene and the expression of each targeted gene was analyzed by 2^−ΔΔCT method. Moreover, the control group was used as a calibrator sample and was set as 1× expression of each targeted gene. Three independent experiments were performed.

Total RNAs were isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed using the RevertAid™ First-Strand cDNA Synthesis Kit (Fermentas, Inc., Hanover, MD, USA). Real-time RT-PCR was performed using the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). PCR amplifications were performed using the SYBR Green I qPCR kit (TaKaRa, Shiga, Japan) with specific primers: 5′-CCCCATCTCTGCTCACTGTC-3′ and 5′-TTTTTCTGGACCGCATTG-3′ for mouse LCN2 (GenBank: NM_008491), and 5′-TGACCACAGTCCATGCCATC-3′ and 5′-GACGGACACATTGGGGGTAG-3′ for mouse Gapdh (GenBank: NM_001289726).