RNA Profiling on the Affymetrix Genetitan Platform
RNA Profiling on the Affymetrix GeneTitan Platform
Contents
Section A: Whole-Transcript (WT) Gene-ST Array Plates:
Section B: miRNA 4.1 Array Plates:
Section C: Gene-ST and miRNA Expression Data Analysis
Section A: Whole-Transcript (WT) Gene-ST Array Plates:
On the GeneTitan you can run Gene-ST, Whole-Transcript (WT) array plates for around 20 different species of model and applied research organisms, in addition to Human, Mouse and Rat.
Instead of a fixed number of probes towards the 3’ end of genes, these arrays have probes corresponding to individual exons, so that the gene expression signal is derived from probes positioned along the whole length of the gene (average ~25 probes). They are unique in that you can also analyse these arrays at the exon-level as well as the gene-level, and so derive additional information about expression of alternative isoforms.
The Human, Mouse and RatGeneST arrays provide updated and comprehensive content (including >11,000 lncRNAs).
For example, Human GeneST2.1 Whole-Transcript array plates:
902136Human Gene 2.1 ST 16-Array Plate and TraysConsists of 16 arrays
902137Human Gene 2.1 ST 24-Array Plate and TraysConsists of 24 arrays
902138Human Gene 2.1 ST 96-Array Plate and TraysConsists of 96 arrays
Note: For Human, Mouse and Rat, Gene 2.1 ST array plates are more up to date and supersede the Gene 1.1 ST array plates.
Further information is available on the website at:
Human Gene 2.1 ST:
MouseGene 2.1 ST:
Rat Gene 2.1 ST:
Each array plate is supplied with a full set of staining and scanning trays for processing in the GeneTitan; these do not need to be ordered separately.
WT Array RNA Sample Preparation and Labelling: Two Options:
1. Standard Method: GeneChip® WT PLUS Reagent Kit:
Target preparation (cDNA synthesis, amplification & labelling) from 50ng-500ng intact totalRNA:
902280GeneChip® WT PLUS Reagent Kitfor 10 RNA samples
902281GeneChip® WT PLUS Reagent Kitfor 30 RNA samples
2. WT-Pico Kit: Sub-Nanogram and FFPE Extracted RNA
Sample Preparation for Whole-Transcriptome (WT) arrays from as little as 100pg TotalRNA (equivalent to approx. 10 cells). Recommended starting amount is 500pg-10ng.
For total RNA purified from Formalin-Fixed, Paraffin Embedded Tissues (FFPE) the recommended starting amount is 500pg to 50ng RNA.
902622GeneChip® WT Pico Kitfor 12 RNA samples
902623GeneChip® WT Pico Kit for 30 RNA samples
For each WT expression array plate you will need the following Genetitan Reagent Kits:
901622GeneTitan® Hybridization, Wash, and Stain Kit for WT Array Platesfor 96 RNA samples
901622 consists of two different modules:
901621:GeneTitan® Hybridization Module for WT Array Plates. Hybridization and staining reagents for 6x16 reactions, 4x24 reactions, or 1x96 reactionsN.B. 901621 is not available for purchase separately.
901583GeneTitan® Wash Buffers A and B Module.1 x 901583 required for each array plate, whether a 16, 24 or 96 array plate.
901583 is available separately and is required for each array plate to be processed (16, 24 or 96).
So in other words, this means 1x901622 for is all that is required for 96 RNAs run on a single 96-array plate, but you will need 1 x 901622 plus additional 3 x 901583 for 96 RNAs run on 4x24-array plates, and 1 x 901622 plus 5 x 901583 for 96 RNAs run on 6x16-array plates.
Section B: miRNA 4.1 Array Plates:
With the GeneTitan you can also run miRNA array plates, to assay small non-coding RNAs from miRBase20 (miRNA, snoRNA, and scaRNA) for 203 different species. The latest version is the miRNA4.1 array.
For miRNA analysis, there are 16, 24 and 96 array plates available, recently updated to interrogate all mature miRNA sequences in miRBase Release 20:
902408Affymetrix miRNA 4.1 16-Array Plate and TraysConsists of 16 arrays
902409Affymetrix miRNA 4.1 24-Array Plate and TraysConsists of 24 arrays
902410Affymetrix miRNA 4.1 96-Array Plate and TraysConsists of 96 arrays
For each miRNA array plate experiment you will require the following:
- 902276 GeneTitan Hyb, Wash and Stain Kit for miRNA Array Platesfor 96 RNA samples
902276 consists of two different modules:
902275GeneTitan Hybridization and Stain Module for miRNA Array Plates– sufficient for 6x16 reactions, 4x24 reactions, or 1x96 reactions
901583GeneTitan® Wash Buffers A and B Module – 1 required for each array plate, whether a 16, 24 or 96 array plate.
So similar to the WT array plates, 1x902276 for is all that is required for 96 RNAs run on a single 96-miRNA array plate, but you will need 1 x 902276 plus additional 3 x 901583 for 96 RNAs run on 4x24-miRNA array plates, and 1 x 902276 plus 5 x 901583 for 96 RNAs run on 6x16- miRNA array plates.
2. 900454GeneChip Hybridization Control Kitfor 30 arrays
3. Affymetrix® FlashTag™ Biotin HSR RNA Labeling Kits: Target Preparation for the miRNA arrays (RNA labelling; no enrichment or amplification is required) in two sizes:
901910FlashTag™ Biotin HSR RNA Labeling Kitsfor 10 RNA samples
901911FlashTag™ Biotin HSR RNA Labeling Kitsfor 30 RNA samples
Section C: Gene-ST and miRNA Expression Data Analysis
There are two RNA expression data analysis packages free to download from the Affymetrix website.
Together they provide the complete workflow for expression profiling from raw image data (CEL) files through to Differential Gene Expression (DGE) analysis and filtering.
The Expression Console (EC) software is free to download from the website at:
Expression Console is a simple, windows package, for RMA based probe-set summarization/normalization and for array QC. With EC you can generate gene-level & exon level expression signals, then generate QC reports, and plot various charts to assess array performance e.g. Signal histogram, Box plots, Pearson Correlation and PCA.
After array QC, the next steps are to perform Differential Gene Expression (DGE) analysis and transcript filtering between experimental groups. These can be performed with the Affymetrix Transcriptome Analysis Console (TAC) software is free to download at:
The most recent version of TAC includes visualization of Gene pathway networks, and combines data from gene expression and miRNA arrays to generate miRNA and target gene interaction plots.
Mar 2017