Total RNA was isolated from the cells using Nucleospin RNA (Mackerey-Nagel, Germany), according to manufacturer’s instructions. Total RNA concentration was measured using the Nanodrop spectrophotometer and then used for reverse transcription using SuperScript III Reverse Transcriptase (Invitrogen, Waltham, MA). A screening of effects on oxidative stress pathway genes was performed using a Qiagen RT2 Profiler PCR array focused on Human Oxidative Stress (PAHS-065Y). Further validation was performed with primers purchased from Qiagen (Valencia, CA). PCRs were performed using RT2 SYBR Green ROX FAST Mastermix (Qiagen), in a 7900HT Fast Real-Time PCR System with Fast 96-Well Block Module (Applied Biosystems, Foster City, CA), with a SDS Plate utility v2.2 software (Applied Biosystems). The results were normalized to the average expression of GAPDH and 18S.
7900ht fast real time pcr system with fast 96 well block module
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 7900HT Fast Real-Time PCR System with Fast 96-Well Block Module is a compact, high-throughput real-time PCR instrument designed for efficient nucleic acid analysis. It features a Fast 96-Well Block Module that enables rapid thermal cycling for accelerated data acquisition. The system is capable of performing real-time PCR experiments with fluorescent-based detection.
Automatically generated - may contain errors
Lab products found in correlation
Sds plate utility v2, by Thermo Fisher Scientific (5 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (5 mentions)
Rt2 sybr green rox fast mastermix, by Qiagen (3 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Rt sybr green rox fast mastermix, by Qiagen (2 mentions)
Rt2 profiler pcr array, by Qiagen (1 mentions)
Rnaeasy mini kit, by Qiagen (1 mentions)
5 protocols using 7900ht fast real time pcr system with fast 96 well block module
1
Oxidative Stress Pathway Gene Expression
2
Gene Expression Validation Protocol
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Validation of gene array data was performed both in the same samples and in two additional independent experiments. RNA was reverse transcribed using SuperScript III Reverse Transcriptase (Invitrogen, Waltham, MA). Most primers were purchased from Qiagen (Valencia, CA). PCRs were performed using RT2 SYBR Green ROX FAST Mastermix (Qiagen), in a 7900HT Fast Real-Time PCR System with Fast 96-Well Block Module (Applied Biosystems, Foster City, CA) with a SDS Plate utility v2.2 software (Applied Biosystems). The results were normalized to the expression of GAPDH.
3
Quantitative RT-PCR Analysis of Gene Expression
4
Gene Expression Analysis with qPCR
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Gene expression validations of interest were performed on RNA from biological replicates, extracted using Qiagen kits, and reverse transcribed using SuperScript III Reverse Transcriptase (Invitrogen, Waltham, MA, USA). Primers for RT2 qPCR were purchased from Qiagen (Valencia, CA, USA), for SIRT1 (catalog number PPH02188A-200) and APP (catalog number PPH05947A-200). PCRs were performed using RT² SYBR Green ROX FAST Mastermix (Qiagen) in a 7900HT Fast Real-Time PCR System with Fast 96-Well Block Module (Applied Biosystems, Foster City, CA, USA) with an SDS Plate utility v2.2 software (Applied Biosystems). The results were normalized to the geometric mean of both glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 18S ribosomal RNA housekeeping genes, with PrimeTime qPCR assay oligomers (catalog numbers Hs.PT.39a.22214836, and Hs.PT.39a.22214856, respectively, Integrated DNA Technologies, San Diego, CA, USA).
5
Quantifying Dopamine Receptor Expression
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Total RNA was isolated from the cells using RNAeasy Mini kit (Qiagen), according to the manufacturer’s instructions. Total RNA concentration was measured using the Nanodrop spectrophotometer and then used for reverse transcription using SuperScript III Reverse Transcriptase (Invitrogen, Waltham, MA). Most primers were purchased from Qiagen (Valencia, CA). PCRs were performed using RT2 SYBR Green ROX FAST Mastermix (Qiagen), in a 7900HT Fast Real-Time PCR System with Fast 96-Well Block Module (Applied Biosystems, Foster City, CA), with a SDS Plate utility v2.2 software (Applied Biosystems). The results were normalized to the expression of GAPDH. The primers for the DA receptors were previously described [18 (link)]. Primers for CCR5 were purchased from Qiagen.
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