Total RNA was isolated from snap frozen tissue and cells using Qiashredder and Qiazol Lysis Buffer on Qiacube-HT following the RNeasy 96 QIAcube HT total RNA cell with DNase protocol according to manufacturer’s instructions (Qiagen). Reverse transcription was performed from 50 ng of total RNA (Thermo Scientific #4374967) and genes of interest were pre-amplified (Thermo Scientific #4488593; 14 cycles) using a pool of TaqMan primers (listed in Additional file 8 : Table S3), following the manufacturer’s instructions (Thermo Scientific), and further run on a 96.96 Fluidigm Dynamic array on the Biomark according to the manufacturer’s instructions (Fluidigm). Data was collected and analyzed using Fluidigm Real-Time PCR Analysis 2.1.1 providing Ct values. All gene expression calculations were performed in Jmp®13.0.1, and data represented in TIBCO Spotfire® 6.5.2 or GraphPrism®. Ct values were normalized to the average of housekeeping genes (dCt), and all treatment group compared to the average control group (-ddCt) and Fold Change was calculated by taking 2^-ddCt. Statistical analysis of gene expression data (-ddCt) was performed in Jmp®13.0.1, using a pairwise Student’s t-test, which identifies genes significantly modulated compared to control. GSVA scoring [32 (link)] was performed using genes defined in Rooney et al. [33 (link)] (Additonal file 8 : Table S3).
Fluidigm real time pcr analysis 2
Manufactured by Standard BioTools
Fluidigm Real-Time PCR Analysis 2.1.1 is a software application for real-time PCR data analysis. It provides tools for managing sample information, visualizing amplification curves, and analyzing gene expression data.
Automatically generated - may contain errors
3 protocols using fluidigm real time pcr analysis 2
1
Quantitative Gene Expression Analysis
2
Gene Expression Analysis of Snap-Frozen Tissues
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Total RNA was isolated from snap frozen tissue and cells using Qiashredder and Qiazol Lysis Buffer on Qiacube-HT following the RNeasy 96 QIAcube HT total RNA cell with DNase protocol according to manufacturer's instructions (Qiagen). Reverse transcription was performed from 50ng of total RNA (Thermo Scientific #4374967) and further pre-amplified (Thermo Scientific #4488593; 14 cycles) using a pool of TaqMan primers (listed in Table S1), following the manufacturer's instructions (Thermo Scientific), and further run on a 96.96 Fluidigm Dynamic array on the Biomark according to the manufacturer's instructions (Fluidigm). Data was collected and analysed using Fluidigm Real-Time PCR Analysis 2.1.1 providing Ct values. All gene expression calculations were performed in Jmp®13.0.1, and data represented in TIBCO™ Spotfire® 6.5.2 or GraphPrism®. Ct values were normalised to the average of housekeeping genes (dCt), and all treatment group compared (subtracted) to the average control group (-ddCt) and Fold Change was calculated by taking 2-ddCt. Statistical analysis of gene expression data (-ddCt) was performed in Jmp®13.0.1, using a pairwise Student's t-test, which identify genes significantly modulated compared to control.
3
Comprehensive RNA Isolation and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Total RNA was isolated from snap-frozen tissue and cells using Qiashredder and Qiazol Lysis Buffer on Qiacube-HT following the RNeasy 96 QIAcube HT total RNA cell with DNase protocol according to the manufacturer's instructions (Qiagen). Reverse transcription was performed from 50 ng of total RNA (Thermo Fisher Scientific #4374967) and genes of interest were preamplified (Thermo Fisher Scientific #4488593; 14 cycles) using a pool of TaqMan primers (listed in Supplementary Table S1), following the manufacturer's instructions (Thermo Fisher Scientific), and further run on a 96.96 Fluidigm Dynamic array on the Biomark according to the manufacturer's instructions (Fluidigm). Data were collected and analyzed using Fluidigm Real-Time PCR Analysis 2.1.1 providing Ct values. All gene expression calculations were performed in Jmp13.0.1, and data represented in TIBCO Spotfire 6.5.2 or GraphPrism. C t values were normalized to the average of housekeeping genes (dC t ), and all treatment group compared with the average control group (ÀddC t ) and fold Change was calculated by taking 2^ÀddC t . Statistical analysis of gene expression data (ÀddC t ) was performed in Jmp13.0.1, using a pairwise Student t test, which identify genes significantly modulated compared with control. GSVA scoring (19) was performed using genes defined in Rooney and colleagues (20) .
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