RNA was extracted from cell pellets using RNeasy Mini Kit with on-column DNase digestion (Qiagen; Hilden, Germany). cDNA synthesis was performed as described in the Expression Analysis Technical Manual (Affymetrix; Santa Clara, CA). GAPDH was used as control gene and related gene expression (Bcl-2, Mcl-1, Bcl-xL, BID, and BAX), (all from IDT, Coralville, Iowa) was analyzed on an Applied Biosystems 7500 Real-Time PCR System using Taqman Universal PCR Master Mix #4304437 and Assay on Demand primer/probe kits (Applied Biosystems; Waltham, MA). For TLDA assay, TLDA v2.0 was performed on the 7900HT real-time PCR system (Applied Biosystems) according to the manufacturer’s protocol. Average delta CT was acquired from the results for further analysis. PCR cycling conditions were performed as follows: 95 °C for 15 s and 60 °C for 1 min, 40 cycles and then 95 °C for 10 min. To normalize RNA input, Human RNU44 small RNA was used as an internal control.
Expression analysis technical manual
Manufactured by Thermo Fisher Scientific
The Expression Analysis Technical Manual provides detailed information and guidelines for conducting expression analysis experiments. The manual covers topics such as sample preparation, data analysis, and interpretation of results, serving as a comprehensive reference for researchers and scientists working in the field of gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fluidics station 450, by Thermo Fisher Scientific (3 mentions)
Genechip scanner 3000 7g, by Thermo Fisher Scientific (3 mentions)
Gapdh, by Thermo Fisher Scientific (2 mentions)
Mcl 1, by Thermo Fisher Scientific (2 mentions)
Taqman universal pcr master mix 4304437, by Thermo Fisher Scientific (2 mentions)
Bcl 2, by Thermo Fisher Scientific (2 mentions)
Bcl2 associated x (bax), by Thermo Fisher Scientific (2 mentions)
Bcl xl, by Thermo Fisher Scientific (2 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
7900ht real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Genechip operating software gcos, by Thermo Fisher Scientific (2 mentions)
Gene chip 133 plus 2, by Thermo Fisher Scientific (2 mentions)
Fluidics station, by Thermo Fisher Scientific (2 mentions)
Genespring gx 12, by Agilent Technologies (2 mentions)
Hgu133 plus 2.0 microarrays, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
10 protocols using expression analysis technical manual
1
Quantitative Analysis of Apoptosis-Related Genes
2
Transcriptomic Profiling of Diabetic Nephropathy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human renal biopsies from patients with diabetic nephropathy (DN) (n = 7) and livinv donor (LD) controls (n = 18) were collected within the framework of the European Renal cDNA Bank—Kröner-Fresenius Biopsy Bank (24 (link)). Biopsies were obtained from patients after informed consent and with approval of the local ethics committees. Following renal biopsy, the tissue was transferred to RNase inhibitor and microdissected into glomerular and tubular fragments. Total RNA was isolated from both micro-dissected compartments, linearly amplified and hybridized to Affymetrix HG-U133 Plus 2.0 microarrays as reported previously (25 (link)). Fragmentation, hybridization, staining, and imaging were performed according to the Affymetrix Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA). The raw data was normalized using Robust Multichip Algorithm (RMA) and annotated by Human Entrez Gene custom CDF annotation version 18 (http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/genomic_curated_CDF.asp ). To identify differentially expressed genes the SAM (Significance analysis of Microarrays) method was applied using TiGR (MeV, Version 4.8.1) (26 (link)). A q-value below 5% was considered to be statistically significant.
3
Total RNA Extraction and Microarray Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using Trizol reagent (Invitrogen). Probe preparation and hybridization, staining and washing of the Affymetrix high-density arrays were carried out as described in the Expression Analysis Technical Manual (Affymetrix).
4
Affymetrix Microarray Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Biotinylated cRNA was prepared according to the standard Affymetrix protocol from 200 ng of total RNA (Expression Analysis Technical Manual, 2001; Affymetrix). Following fragmentation, 3 μg of RNA were hybridized for 16 hours at 45°C on the GeneChip® Human Genome Array. The GeneChip® was washed and stained in the Affymetrix Fluidics Station 450. The GeneChip® was scanned using the Affymetrix GeneChip® Scanner 3000 7G. The data were analyzed with Robust Multichip Analysis using the Affymetrix default analysis settings and global scaling as the normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100. The normalized and log transformed intensity values were then analyzed using GeneSpring GX 12.6.1 (Agilent Technologies, Santa Clara, CA, USA). Fold change filters included the requirement that the genes be present in at least 150% of the controls for upregulated genes and fewer than 66% of the controls for downregulated genes. Through hierarchical clustering, data were clustered into groups that behaved similarly across experiments using GeneSpring GX 12.6.1 (Agilent Technologies). The clustering algorithm used the Euclidean distance with average linkage.
5
AGO1 IP and Drosophila Transcriptome
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The samples were collected at 18°C, 25°C, or 29°C. AGO1 immunoprecipitation was performed as previously described (Kadener et al., 2009 (link); Lerner et al., 2015 (link)). Input and AGO1 IP Total RNA was extracted from fly heads using TRI reagent (Sigma) according to manufacturer’s protocol. cDNA synthesis was carried out as described in the Expression Analysis Technical Manual (Affymetrix). The cRNA reactions were carried out using the IVT kit (Affymetrix). Affymetrix high-density arrays for D. melanogaster version 2.0 were probed, hybridized, stained, and washed according to the manufacturer’s protocol.
6
Genome-wide Transcriptome Analysis by Microarray
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
15ug of fragmented biotinylated cRNA was hybridized to Gene Chip Human Genome U133 Plus 2.0 Array. Following standard conditions described in Affymetrix protocol (Expression Analysis Technical Manual, Affymetrix, Santa Clarita, CA). Briefly, fragmented biotinylated cRNA was incubated with Gene Chip 133 plus 2.0 for 16 hours at 45oC before the hybridized chip was washed and stained in an Affymetrix fluidics station. The processed array was scanned, and the Q.C. was performed using Gene Chip Operating Software (GCOS, Affymetrix, CA). Same specimens performed by RNA-seq will be reported separately (although all data from single-cell RNA-seq will be presented by other manuals).
7
RNA Extraction and Real-Time PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 11
RNA was extracted from cell pellets using RNeasy Mini Kit with on-column DNase digestion (Qiagen; Hilden, Germany). cDNA synthesis was performed as described in the Expression Analysis Technical Manual (Affymetrix; Santa Clara, Calif.). GAPDH was used as control gene and related gene expression (Bcl-2, Mcl-1, Bcl-xL, BID and BAX; all from IDT, Coralville, Iowa), was analyzed on an Applied Biosystems 7500 Real-Time PCR System using Taqman Universal PCR Master Mix #4304437 and Assay on Demand primer/probe kits (Applied Biosystems; Waltham, Mass.). For TLDA assay, TLDA v2.0 was performed on the 7900HT real-time PCR system (Applied Biosystems) according to the manufacturer's protocol. Average delta CT was acquired from the results for further analysis. PCR cycling conditions were performed as follows: 95° C. for 15 s and 60° C. for 1 min, 40 cycles and then 95° C. for 10 min. To normalize RNA input, Human RNU44 (Table 4) small RNA was used as an internal control.
8
Affymetrix Gene Expression Profiling Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Biotinylated cRNA samples were prepared according to the standard Affymetrix protocol from 500 ng total RNA (Expression Analysis Technical Manual, 2001, Affymetrix). Following fragmentation, 15 mg of aRNA was hybridized for 16 h at 45 °C on a GeneChip Mouse Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450. GeneChips were scanned using the Affymetrix GeneChip 3000 7G scanner. The data were analyzed with RMA using Affymetrix default analysis settings with global scaling as the normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100. The normalized and log-transformed intensity values were then analyzed using GeneSpring GX 12.6 (Agilent Technologies, Santa Clara, CA, USA). Fold change filters included the requirement that the genes be present in at least 200% of controls for upregulated genes and lower than 50% of controls for downregulated genes. Hierarchical clustering data were clustered groups that behaved similarly across experiments using GeneSpring GX 12.6 (Agilent Technologies). The clustering algorithm involved Euclidean distance and average linkage.
9
Genome-wide Transcriptome Analysis by Microarray
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
15ug of fragmented biotinylated cRNA was hybridized to Gene Chip Human Genome U133 Plus 2.0 Array. Following standard conditions described in Affymetrix protocol (Expression Analysis Technical Manual, Affymetrix, Santa Clarita, CA). Briefly, fragmented biotinylated cRNA was incubated with Gene Chip 133 plus 2.0 for 16 hours at 45oC before the hybridized chip was washed and stained in an Affymetrix fluidics station. The processed array was scanned, and the Q.C. was performed using Gene Chip Operating Software (GCOS, Affymetrix, CA). Same specimens performed by RNA-seq will be reported separately (although all data from single-cell RNA-seq will be presented by other manuals).
10
Microarray Analysis of Aging and Mutation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Biotinylated cRNA were prepared from 250 ng total RNA according to the standard Affymetrix protocol (Expression Analysis Technical Manual, 2001, Affymetrix). Following fragmentation, 15 μg of RNA were hybridized for 16 hr at 45°C on a GeneChip Mouse Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450. GeneChips were scanned using the Affymetrix Gene Chip Scanner 3000 7G. Eight microarray samples were normalized by the RMA method of R affy package [41 (link)]. DEG testing was accomplished using the 4 class limma test of 2 mutants and 2 aging factors. Finally, 2,197 probes were considered to have significantly differential expression by a cut-off p-value < 0.05.
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!