Total RNA was extracted from the hypoxia-exposed and si-RNA treated cells, and equal amounts (2 μg) were subsequently transcribed into cDNA using M-MuLV reverse transcriptase (Thermo Fisher Scientific). The cDNA was used as a template in real-time PCR performed with the ABI Prism 7300 detection system (Applied Biosystems, Foster City, CA, USA) with SYBR green as a fluorescent dye (Invitrogen). Human-specific primers for PKM2, HIF-1α, HIF-2α, VEGF, and PHD3 (sequences shown in S2 Table ) were designed using sequence information from NCBI and were purchased from Biomers (Biomers). Expression was analysed by the ΔCt method. The Ct values of the target genes were normalized to that of the porphobilinogen deaminase (PBGD) gene (endogenous control).
Abi prism 7300 detection
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI Prism 7300 is a real-time PCR detection system. It is designed to provide accurate and reproducible results for gene expression analysis, SNP genotyping, and other quantitative PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3 first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
M mulv reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Taqman universal master mix, by Thermo Fisher Scientific (1 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Total rna purification kit, by Norgen Biotek (1 mentions)
Sybr green pcr reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using abi prism 7300 detection
1
Quantifying Hypoxia-Induced Gene Expression
2
Quantitative Real-Time PCR Analysis of U251 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was isolated from U251 human glioma cells before and after the induction using Trizol reagent (Invitrogen, Carlsbad, CD, USA). cDNA was transcribed using superscript III first strand cDNA synthesis kit following the manufacturer's instructions (Invitrogen, Carlsbad, CD, USA). Quantitative real-time PCR was performed with SYBR Green PCR reagents on an ABI Prism 7300 detection system (Applied Biosystems, Foster City, CA). Beta-actin was used as an internal control. The normalized fold expression was obtained using the 2−ΔΔCT method. Primers used for real-time PCR are summarized in Table 1 .
3
Quantifying Gene Expression in Breast Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from frozen MCF-7, T47D and HMLE cell pellets (−80°C) with Total RNA purification kit (Norgen Biotek Corp.), according to the manufacturer's instructions. DNAse treatment was performed using TURBO DNA-free kit (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. cDNA synthesis was performed with the SuperScript™ VILO™ cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. RT-qPCR was performed with ABI Prism 7300 detection apparatus (Applied Biosystems; Thermo Fisher Scientific, Inc.) using Taqman Universal Master Mix according to the manufacturer's recommendations. Cq value was determined with Sequence Detection System software. All primers were from Applied Biosystems (Thermo Fisher Scientific, Inc.; Appendix S1 ). Levels of gene expression were determined using GENORM software and normalized using GAPDH and RPLPO.
4
Quantitative Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was transcribed using Superscript III First Strand cDNA Synthesis kit following the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Quantitative real-time polymerase chain reaction (PCR) was performed with SYBR Green PCR reagents on an ABI Prism 7300 detection system (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The normalized fold expression was obtained using the 2-ΔΔ CT method. Primers used for real-time PCR are shown in Table 1 .
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!