RNA from HNC cells was extracted using Trizol (Invitrogen, Life Technologies, Carlsbad, CA), purified by RNA-cleanup (Qiagen, Gaithersburg, MD), Random hexamers, MuLV enzyme was used for cDNA synthesis (Applied Biosystems, Foster City, CA). PCR pre-developed probe for SHP2, HLA-B, STAT1, TAP-1 and β-actin were purchased from Applied Biosystem for TaqMan® Gene Expression Assay. Real-time PCR (7700 Real-Time PCR System; Applied Biosystems, Carlsbad, CA) used the following conditions: denaturation at 95°C for 10s, annealing at 60°C for 15s, and extension at 72°C for 30s. An initial denaturation step at 95°C for 5 min and final extension step at 72°C for 10 min were also included. Relative expression of the gene to endogenous control gene (β-actin/GUS) was calculated using the ΔCT method: relative expression = 2−ΔCT, where ΔCT = CT (shp2) − CT (β-actin/gus).
7700 real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 7700 Real-Time PCR System is a laboratory equipment used for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. It is designed to perform precise and accurate quantification of DNA or RNA targets in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Rna cleanup, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Quantifast sybr green pcr kit, by Qiagen (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Merck Group (1 mentions)
Sds software package, by Thermo Fisher Scientific (1 mentions)
3 protocols using 7700 real time pcr system
1
Quantitative Real-Time PCR Analysis of Gene Expression
2
Total RNA Isolation and HSP90A Quantification
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Total RNA from cells was isolated in 2 steps using Trizol reagent (Invitrogen, MA, USA) followed by RNeasy (Qiagen, Valencia, CA, USA) purification, then subjected to reverse transcription. HSP90A was amplified with primers (5′-CCTTCTATTTGTCCCACG-3′,5′-AGATCCTCCGAGTCTACCAC-3′) and conditions (40 cycles, 95°C for 10 min, 95°C for 15 s, and 60°C for 1 min). GADPH was used as an internal control, ensuring cDNA quality and loading accuracy. All samples were run in duplicate on a 7700 Real-Time PCR System (Applied Biosystems, MA, USA) and the data were analyzed using Sequence Detector v1.9 software.
3
Quantitative Gene Expression Analysis
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Expression analyses were performed using QuantiFast Sybr Green PCR kit (Qiagen, Milano, Italy), following the one-step protocol: cDNA was first synthesized from 60 ng total RNA, and then amplified by means of the Sybr Green QuantiTect Primer (Qiagen, Milano, Italy): hENT1 (QT010000083), CHOP (QT00082278), MRP1 (QT00061159) and DKC (QT00000392). Reactions were set up in 96-well plates and loaded onto 7700 Real-Time PCR System (Applied Biosystems, Foster City, CA). Optical data obtained were analyzed using the SDS software package (version 1.9.1; Applied Biosystems, Foster City, CA). Expression levels of target gene were obtained using the comparative method of relative quantification, after normalization for the housekeeping control gene Glyceraldehyde-3-phosphate dehydrogenase GAPDH (Sigma Aldrich, Milano, Italy), as previously performed [20 (link)].
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