RNA was isolated from cultured cells or from cancerous and benign tissue sections, as marked by the study pathologist, of an unbaked FFPE slide using the FFPR RNA easy kit (Qiagen), as we have done previously [13 ]. RNA was reverse transcribed and relative target-gene expression was assessed by quantitative-PCR (qPCR) with a SYBR green detection dye (Invitrogen) and Rox reference dye (Invitrogen) on the Step One Real Time PCR System (Applied Biosystems). Using the ΔΔCt relative quantification method, target gene readouts were normalized to RPL19 and GADPH transcript levels. Experiments were the average of biological triplicates. Target genes included AR and a 20-gene panel that has been previously used to define an “AR activity score;” [14 ] this 20 gene panel itself was derived from previously published AR activity gene panels [15 ,16 ]. We found that 12 of these 20 genes (FKBP5, MED28, ELL2, KLK2, PMEPA1, CENPN, C1ORF116, NKX3.1, KLK3, EAF2, TMPRSS2, HERC3) were robustly expressed and androgen-induced in the six ovarian cancer cell lines. The transcript levels, relative to housekeeping genes, were compared to those from LNCaP cells, a prototypical prostate cancer cell line with robust AR activity, and the mean percent of LNCaP transcript levels was reported as the AR activity score.
Sybr green detection dye
Manufactured by Thermo Fisher Scientific
SYBR green detection dye is a fluorescent dye used in molecular biology applications to detect and quantify DNA. It binds to double-stranded DNA, emitting a green fluorescent signal upon excitation. The dye is commonly used in real-time PCR (qPCR) experiments to monitor the amplification of specific DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Rox reference dye, by Thermo Fisher Scientific (3 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (3 mentions)
Ffpr rna easy kit, by Qiagen (2 mentions)
Moloney murine leukemia virus reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (1 mentions)
3 protocols using sybr green detection dye
1
Quantifying AR Activity in Ovarian Cancer
2
Quantifying AR Activity in Ovarian Cancer
3
Quantitative Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The total RNA was isolated from the cells
using the GeneJET RNA purification kit (Thermo Scientific). The isolated
RNA was then reverse-transcribed with Moloney murine leukemia virus
reverse transcriptase (Invitrogen). The relative target gene expression
was then assessed by qPCR with an SYBR green detection dye (Invitrogen)
and Rox reference dye (Invitrogen) on the StepOne Real-Time PCR System
(Applied Biosystems). Using the ΔΔCt relative quantification method, the target
gene readouts were normalized to RPL19 and GADPH transcript levels.
The experiments are the average of biological triplicates; p values were calculated using a two-tailed Student’s t- test.
using the GeneJET RNA purification kit (Thermo Scientific). The isolated
RNA was then reverse-transcribed with Moloney murine leukemia virus
reverse transcriptase (Invitrogen). The relative target gene expression
was then assessed by qPCR with an SYBR green detection dye (Invitrogen)
and Rox reference dye (Invitrogen) on the StepOne Real-Time PCR System
(Applied Biosystems). Using the ΔΔCt relative quantification method, the target
gene readouts were normalized to RPL19 and GADPH transcript levels.
The experiments are the average of biological triplicates; p values were calculated using a two-tailed Student’s t- test.
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