Total RNA was isolated by using the AxyPrepTM Multisource Total RNA Miniprepkit (Axygen, USA). An equivalent amount of RNA was converted into complementary DNA (cDNA) with PrimeScript™ RT reagent Kit (Takara,Japan). Subsequently, Real-time PCR was performed using an ABI 7500 Sequencing Detection System and SYBR®Premix Ex Taq (Takara, Japan). All of the procedures were performed according to the manufacturer’s protocols. Cycling condition was as follows: 40 cycles at 95°C for 5 seconds and 60°C for 34 seconds. The primer sequences are, for human Notch1: forward 5′-GGA GGC ATC CTA CCC TTT TC-3′, and reverse 5′-TGT GTT GCT GGA GCA TCT TC-3′; for human HES1: forward 5′-CTC TCT TCC CTC CGG ACT CT-3′ and reverse 5′-AGG CGC AAT CCA ATA TGA AC-3′; for human Caspase3: forward 5′-TTT TTC AGA GGG GAT CGT TG-3′ and reverse 5′-CGG CCT CCA CTG GTA TTT TA-3′; for human Caspase8: forward 5′-TGC AGG GTC TCA CTC TGT TG-3′ and reverse 5′-CAA AAA TCA GCC ATG TGT GG-3′; for human Caspase9: forward 5′-CTA GTT TGC CCA CAC CCA GT-3′; and for human GAPDH: forward 5′-CCT GCA CCA CCA ACT GCT TA-3′ and reverse 5′-AGG CCA TGC CAG TGA GCT T-3′. The comparative 2-ΔΔCT method was used to calculate the relative expression level of each target gene with GAPDH as the housekeeping gene.
Abi 7500 sequencing detection system
Manufactured by Takara Bio
Sourced in Japan
The ABI 7500 Sequencing Detection System is a real-time PCR instrument designed for accurate and reliable DNA sequence detection. It features a 96-well format and utilizes a high-performance optical system to provide precise measurements of fluorescence signals during the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Sybr premix ex taq, by Takara Bio (5 mentions)
Primescript rt reagent kit, by Takara Bio (4 mentions)
Axyprep multisource total rna miniprep kit, by Corning (4 mentions)
Sybr green kit, by Takara Bio (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
6 protocols using abi 7500 sequencing detection system
1
Quantitative Analysis of Notch, Caspase Genes
2
Quantitative Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Large disks were used in this test. First, the disks were placed at the bottom of 24-well plates and ~2×104 cells were seeded on the surface of each large disk, followed by incubation at 37°C in 5% CO2 and saturated humidity for 7 days. Thereafter, total RNA was isolated using the AxyPrep™ Multisource Total RNA Miniprep Kit (Axygen, Corning, NY, USA), and an equivalent amount of RNA was converted into complementary DNA (complementary deoxyribonucleic acid [cDNA]) with PrimeScript™ RT reagent Kit (Takara, Kusatsu, Japan). Subsequently, real-time PCR was performed using an ABI 7500 Sequencing Detection System and SYBR® Premix Ex Taq (Takara). All the procedures were performed according to the manufacturer’s protocols. The employed cycling condition was 40 cycles at 95°C for 5 s and 60°C for 34 s. The primer sequences are shown in Table 1 . The comparative 2-ΔΔCT method was used to calculate the relative expression level of each target gene with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene.
3
Tec-mediated Gene Expression in OS Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The OS cells (Saos2 and U2OS) were dislodged and suspended in medium, and 1×106 cells were seeded into each well of a six-well plate and treated with 0, 100, 200, and 400 μmol/l Tec for 72 h after adherence. Total RNA was isolated using the AxyPrep Multisource Total RNA Miniprepkit (Axygen, Union City, California, USA). Equivalent amounts of RNA were converted into cDNA using the PrimeScript RT reagent kit (Takara, Shiga, Japan). Real-time PCR was performed using an ABI 7500 Sequencing Detection System and SYBR Premix Ex Taq (Takara). All procedures were performed according to the manufacturer’s protocols. Cycling conditions were 40 cycles of 95°C for 5 s and 60°C for 34 s. Primer sequences are listed in Table 1 .
4
Quantifying Inflammation and Matrix Degradation in IVD
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NP cells were incubated with different concentrations of CY and 1 µg/ml LPS for 24 h. LPS could induce inflammation and matrix degradation in IVD. Total RNA was isolated by the AxyPrep™ Multisource Total RNA Miniprep kit (Axygen Biosciences, Union City, CA, USA). Then 1 µg RNA was converted into complementary DNA (cDNA) with PrimeScript™ RT reagent kit (Takara Bio, Inc., Otsu, Japan). Quantitative PCR was performed using an ABI 7500 Sequencing Detection System and SYBR® Premix Ex Taq™ (Takara Bio, Inc.). Cycling conditions were as follows: 40 cycles at 95°C for 5 sec and 60°C for 34 sec. The primers were used to amplify target genes are listed in Table I . The primers were designed and selected using blast in pubmed, and GAPDH was used as the internal control.
5
RT-qPCR Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was collected using TRIzol Reagent (Takara, Japan). 1 μg of RNA was collected as a template and reversely transcribed using PrimeScript™ RT Master Mix (TaKaRa, Japan). The reversed cDNAs were used for the following RT-qPCR experiments using the SYBR®Green kit (TaKaRa, Tokyo, Japan), and the reactions were performed using an ABI 7500 Sequencing Detection System. The primers used in RT-qPCR were synthesized by GENEWIZ (Suzhou, China). The primer sequences are displayed in Table 1 . With GAPDH as a reference gene, Ct and 2−ΔΔCt values were employed to calculate the mRNA levels.
6
Quantitative RT-PCR Analysis of Osteogenic Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was prepared using Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA) for cellular extracts. cDNA was then generated from 1 μg of RNA using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Quantitative real-time expression analysis was performed using an ABI 7500 Sequencing Detection System and SYBR Premix Ex Taq (Takara, Japan). Relative expression of mRNA was determined after normalization using the ΔCt method. The following primers were used: GAPDH, 5′-ATGGGGAAGGTGAAGGTCG-3′ (forward) and 5′-GGGGTCATTGATGGCAACAATA-3′ (reverse); Runx2, 5′-CCGCCTCAGTGATTTAGGGC-3′ (forward) and 5′-GGGTCTGTAATCTGACTCTGTCC-3′ (reverse); ALP, 5′-TGAGGGTGTGGCTTACCAG-3′ (forward) and 5′-GATGGACGTGTAGGCTTTGCT-3′ (reverse); BSP, 5′-CAGGGAGGCAGTGACTCTTC-3′ (forward) and 5′-AGTGTGGAAAGTGTGGCGTT-3′ (reverse); and Col-1, 5′-GAAAAGGGTACATCGGGTGAG-3′ (forward) and 5′-GAACCCATCGAGTCCTGGT-3′ (reverse).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!