Transcriptome Profiling Support - Getting Started

The Clariom™ S is a 169 format, the correct script is FS450_0007.

No, the Clariom™ S array is not capable of exon-level analysis. The probes have selected to be the most constitutive probes, representing the most common areas of a gene model. This means that most of the exons in the gene model will likely not have representation on the array, meaning that it is not practical to enable exon-level analysis.

Depending on input, sample type, and platform, we have several options: Cartridge: Assays for 100 pg–50 ng of total RNA isolated from whole blood, cultured cells, and fresh/fresh frozen or FFPE tissues.

• Pico Assay Assays for 50–500 ng of total RNA isolated from whole blood, cultured cells, and fresh/fresh frozen tissues.
• WT Plus Assay Plates: Assays for 100 pg–50 ng of total RNA isolated from whole blood, cultured cells, and fresh/fresh frozen or FFPE tissues for analysis on GeneTitan Instrument.
• Pico Assay HT Assays for 50–500 ng of total RNA isolated from whole blood, cultured cells, and fresh/fresh frozen tissues for analysis on GeneTitan Instrument.
• WT Plus HT Assay

No, there is no need to globin reduce your samples prior to starting the assay for the Clariom™ S array.

Clariom™ D Assay scan time is about 33 min

• DAT file size about 790-800MB
• CEL file size about 68MB

Clariom™ S scan time is about 5 min

• DAT file size about 35-40MB
• CEL file size about 3MB

The human, mouse, and rat transcriptome assays are available in three formats based on sample type and amount of input RNA:

• Human, mouse, and rat transcriptome Pico assays compatible with 100–2,000 pg of RNA isolated from fresh, frozen, blood, or FFPE samples
• Human, mouse, and rat transcriptome FFPE assays compatible with 20–200 ng of RNA isolated from FFPE samples
• Human, mouse, and rat transcriptome assays compatible with 50–500 ng of RNA isolated from fresh, frozen, or blood samples

FS450_0001 should be used when processing human, mouse, or rat transcriptome assays. Up-to-date fluidics scripts can be obtained from the website.

Human, mouse, or rat transcriptome arrays are 49-format.

To install library files for the human, mouse, or rat transcriptome arrays for AGCC, simply run the executable library file installer.

Probe cell intensity data (CEL) from human, mouse, or rat transcriptome assays are analyzed in Expression Console Software. The application uses the RMA analysis to create CHP files. Expression Console Software can be downloaded at no charge from the website. Further statistical analysis may be performed in the Transcriptome Analysis Console (TAC) Software to obtain a list of differentially expressed genes and alternative splicing events. TAC Software also provides the visualization of genes, exons, junctions, transcript isoforms, pathways, and mRNA-miRNA regulatory networks. TAC Software can be downloaded at no charge from the website.

Analysis library files and annotation files can be downloaded from within both Expression Console Software and TAC Software. Please refer to the respective user manuals for detailed instructions.

HTAv1, also known as Glue Grant Human Transcriptome Array (GG-H), is a custom array made in collaboration with Stanford Genome Technology, Wing Wong's lab at Stanford, and the Inflammation and Host Response to Injury program ("Glue Grant"). The array was designed to interrogate gene expression, alternative splicing, detection of coding SNPs and non-coding transcription. Permissions from the group are required to use this array. HTA 2.0 is one of our catalog arrays. This array follows a similar design as the HTAv1 but focus on the whole transcript gene expression and alternative splicing of coding and non-coding transcripts. We provide assay and software for the processing and analysis of this array.

The sequences represented on all commercial GeneChip™ expression arrays can be obtained from our website using the NetAffx™ Analysis Center or the annotation and sequence files.

Annotation and sequence files contain the complete entries for all probe sets on the array, taken from the NetAffx™ Analysis Center. They are intended to be used primarily in spreadsheet applications and database programs (such as SQL databases). Interactive and batch queries can be performed in the NetAffx™ Analysis Center to find information for individual probe sets of interest.

Annotation files are available for most GeneChip™ Arrays. They are listed with other available support material on the various array support pages.

Descriptions of Annotation Formats:

MAGE-ML XML Annotations
Manual, Probe Set Data in MAGE-ML Format

CSV Annotations
Manual, Probe Set Data in Tabular Format

Alignments
Manual, Alignments to Genome in PSL Format

The sequences represented on all commercial GeneChip™ expression arrays can be obtained from the NetAffx™ Analysis Center. To search for a gene of interest, go to the Quick Query in the NetAffx™ Analysis Center. You will be required to log in. Highlight the desired array type in #1 and enter the gene name, symbol, probe set ID, or public accession number in #2. Then, click on "Search”.

To search the NetAffx™ Analysis Center using a list of genes, use the Batch Query option in the NetAffx™ Analysis Center. You will be required to log in with your email and password. For your genes of interest, create a tab-delimited text file in Excel or Notepad including gene symbols, probe set IDs, or Accession Numbers (in a column, excluding all header and footer information). Select the desired array type in #1, choose the appropriate search criteria in #2, and upload the .txt file in #3. Now click on "Search."

For all arrays in opaque pouches, customers may anticipate a minimum shelf life of three months after receiving the array. For Tiling arrays in clear pouches customers may anticipate a minimum shelf life of two months after receiving the array.

The Tilling 1.0R Array Set is made up of a set of 14 arrays. The array set contains both perfect match and miss match probes. The 1.0R arrays were designed for transcript mapping experiments. The Tilling 2.0R Array Set is available as a whole genome set (made up of 7 arrays) or individual arrays from the set can be purchased separately. This array is not an updated version of the Tilling 1.0R array. The arrays are made up of only perfect match probes obtained from the Tilling 1.0R array set. The Tilling 2.0R arrays were designed for ChIP experiments. The Promoter 1.0R Array is a single arrays made up of a subset of the probes from the Human 2.0R Array focusing on 7.5 kb upstream through 2.45 downstream of 5’ transcription start sites. The array is made up of perfect match probes.

The NCBI Human Genome Assemply Build 34 was used to design the Human Tilling 1.0R Set, Human Tilling 2.0R Set and the Human Promoter 1.0R Array Repetitive elements were removed by RepeatMasker. Probes were tiled at an average resolution of 35bp as measured from the central position of adjacent 25-mer oligos, leaving a gap of approximately 10bp between probes. Promoter 1.0 Arrays regions were also selected using sequence information from: 35,685 ENSEMBL genes (version 21_34d, May 14, 2004) 25,172 RefSeq mRNAs (NCBI GenBank™, February 7, 2004) 47,062 complete-CDS mRNA (NCBI GenBank™, December 15, 2003)

The NCBI Mouse Genome Assembly version 33 was used to design the Mouse Tilling 1.1R Array set, Mouse Tilling 2.0R Array set, and Mouse Promoter 1.0R Array. Repetitive elements were removed by RepeatMasker. Promoter regions for the Mouse Promoter 1.0R Array were selected using sequence information from: 33,559 ENSEMBL genes (version 30_33f March 23, 2005) 18,167 RefSeq mRNAs (NCBI GenBank™ April 7, 2005) 27,707 complete-CDS mRNAs (NCBI GenBank™ April 7, 2005).

GeneChip™ probe arrays should be stored at 2-8°C, and must not be frozen.

No, Thermo Fisher Scientific does not provide any services for running these arrays. However, several service providers provide GeneChip™ analysis for a fee.

Our experts does not recommend running samples from species other than the one the GeneChip™ expression array was designed for. Although high homology may exist between species, the sequence differences may be sufficient to interfere with hybridization, and more importantly, data interpretation. Although we have not validated the use of the GeneChip™ expression arrays with alternate species, some customers have explored this approach. The following article is an example of this type of study:

Kayo, T., Allison, D.B., Weindruch, R., and Prolla, T.A. Influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkeys. Proceedings of the National Academy of Sciences of the USA. 98:5093-5098, 2001

96 arrays = 4 to 4.6 hours

24 arrays = 1.5 to 2 hours

No, the PM only arrays have probes that are perfect match only. The mismatch probes have been removed. For the HT HGU133 plus PM product roughly ¾ of the probesets have 9 probes, a small portion has 10; the remaining probesets have 11. The Mouse and Rat PM only plate arrays utilize all 11 probes for each probeset.

No, new scripts for Hyb, Wash, and Stain of the HT HMR PM only array plates will not be needed. The script is the same as the old HMR plate arrays.

No, %P is a metric derived using the MAS5 algorithm which has been disabled for the HT PM Plate Arrays.

Run RMA or PLIER in Expression Console 1.1.

• Mean absolute relative log expression – MA (RLE)
• Detection of 20X spikes, Bio B, Bio C, Bio D & Cre
• Housekeeping controls
• Poly A controls
• Antigenomic GC – 12 background probes
• PM mean

Please refer to the QC Metrics for GeneChip™ Human, Mouse and Rat HT PM Array Plates QRC for more information.

The Ambion™ Whole-Transcript (WT) Expression kit, used in conjunction with the Affymetrix™ GeneChip™ WT Terminal Labeling and Controls Reagent kit, is recommended for preparing samples for use with the Gene 1.0 ST Array and the Gene 2.0 ST Array.

Using the Ambion™ WT Expression kit, a minimum of 50 ng of starting total RNA is required. No ribosomal RNA reduction of the starting material is required.

GeneChip™ Operating Software (GCOS) or GeneChip™ Command Console™ Software (AGCC) can be used to process Gene 1.0 ST Arrays through fluidics and scanning. Gene 2.0 ST Arrays utilize an updated installer package that is not compatible with GCOS. Gene 2.0 ST Arrays require AGCC for fluidics and scanning. Affymetrix™ Expression Console™ Software is required to perform QC analysis of Gene 1.0 ST and Gene 2.0 ST Array data. Expression Console Software can be downloaded free from the website.

FS450_00007 should be used when processing Gene 1.0 ST Array. Up-to-date fluidics scripts can be obtained from the website.

FS450_00002 should be used when processing Gene 2.0 ST Array. Up-to-date fluidics scripts can be obtained from the website.

The Gene 1.0 ST Array is a 169 format array.

The Gene 2.0 ST Array is a 100 format array.

Installing the library files for the Gene 1.0 ST Array for AGCC requires the use of the Affymetrix™ Library File Importer which converts GCOS library files into AGCC format, then imports the files to the Command Console library file folder. The Library File Importer can convert files from the local GCOS installation, from a GCOS server on a remote Windows™ Server, or from a GCOS library file package on a CD or disk drive.

To install library files for the Gene 2.0 ST Array for Command Console, simply run the executable library file installer.

For array hybridization, washing, staining and scanning, the following instrumentation is required:

• GeneChip™ Scanner 3000 7G
• Fluidics Station
• Hybridization Oven 640 or Hybridization Oven 645
• Data acquisition and analysis of Mouse 430 2.0 Arrays requires the following software:
• GeneChip™ Command Console Software
• Expression Console Software

A single GeneChip™ Mouse 430 2.0 array generates approximately 130MB of data.

About 12 minutes per GeneChip™ Mouse 430 2.0 Array, comparable to scan time for a set of GeneChip™ Mouse 430A and GeneChip™ Mouse 430B Arrays.

A probe set is a collection of probes designed to interrogate a given sequence. A probe set name is used to refer to a probe set, which looks like the following:

12345_at or 12345_a_at or 12345_s_at or 12345_x_at

The last three characters (_at, in RED) identify the probe set strand. Probe sets that are designed to detect the anti-sense strand of the gene of interest are annotated with "_at".

There are different types of probe sets that can result from the probe selection process. Most probe sets have an extension of an underscore and a letter to designate the probe set type, except for unique probe sets. These different probe set types are shown in the example above in BLUE.

Probes in a gene family probe set (_a set) all cross-hybridize to the same set of sequences that belong to the same gene family (i.e. having same name in the "geneCluster" column). This probe set type is only created if the "geneCluster" column is included in the Instruction File and contains information.

Probes in a unique probe set do not cross-hybridize to any other sequences in the design (including any additional pruning sequences provided).

Probes in an identical probe set (_s set) all cross-hybridize to the same set of sequences that are used for the design (including any additional pruning sequences if provided). These sequences are not defined as from the same gene family for one the following reasons: the values in the "geneCluster" column are different, or the gene family information is not provided.

Probes in a mixed probe set (_x set) contain at least one probe that cross-hybridizes with other sequence(s) used for the design. Cross-hybridizing probes have a cross-hybridization penalty applied to their raw probe scores, and thus, favoring unique probes of the same quality over cross-hybridizing probes.

The following diagram is a graphical representation of these different probe set types.

For example, the line connecting the S1 circle to PS1 indicates that all of the probes in that probe set represent only that particular sequence, whereas the lines connected to PS2 indicate that all probes represent both sequences S2 and S3 in the G1 family (Gene Cluster 1). However, the probes do not represent S4 in the same G1 family. The lines connected to PS3 indicate that all probes in probe set 3 represent both sequences S4 and S5; however the sequences are from different gene clusters (S4 is from G1, S5 is from G2). The lines connected to PS4 indicate that all probes represent S5 in G2; however, one probe (P12) also represents (cross-hybridizes) to sequences S6 and S7.

The GeneChip™ Mouse 430A chip contains primarily probe sets against well annotated full-length genes. The GeneChip™ Mouse 430B chip contains probe sets against gene clusters containing only EST sequences and some gene clusters with non-EST sequences.

Please note, the GeneChip™ Mouse Genome 430 2.0 Array is the recommended replacement for GeneChip™ Mouse 430A and 430B Arrays.

For array hybridization, washing, staining and scanning, the following instrumentation is required:

• GeneChip™ Scanner 3000 7G
• Fluidics Station 450
• Hybridization Oven 640 or Hybridization Oven 645

Data acquisition and analysis of Mouse 430 2.0 Arrays requires the following software:

• GeneChip™ Command Console Software
• Expression Console Software

A single GeneChip™ Rat 230 2.0 array generates approximately 92MB of data.

Scanning a single GeneChip™ Rat 230 2.0 array takes approximately 9 minutes. This is about 25% less time than is required to scan the GeneChip™ Rat 230A and GeneChip™ Rat 230B arrays combined.

Please note, the GeneChip™ Rat Genome 230 2.0 Array is the recommended replacement for GeneChip™ Rat Genome 230A and 230B Arrays.

The GeneChip™ Rat 230A Array contains primarily probe sets against well annotated full-length genes. The GeneChip™ Rat 230B Array contains probe sets against gene clusters containing only EST sequences.

Please note, the GeneChip™ Rat Genome 230 2.0 Array is the recommended replacement for GeneChip™ Rat Genome 230A and 230B Arrays.

Median is 123 bases, and minimum is 25 bases.

PSR is the smallest unit on the exon array for expression profiling and each PSR is represented by an individual probe set. In some cases, each PSR is also an exon; in other cases, due to variation in overlapping exon structures, the PSR can be a subset of the true biological exon. As a result, alternatively spliced exons from the same gene may overlap (i.e., alternative donor or acceptor site); however, PSRs have the property that they do not overlap each other in the genome space, except if annotations change with a newer version of the genome assemblies. In cases where multiple annotations infer different exon structures, that one exon cluster (a group of overlapping exons) will be divided into multiple PSRs. Therefore, in the final design, there are approximately 1,000,000 exon clusters represented by approximately 1,400,000 PSRs.

About 90% of the exon-based probe sets contain 4 probes for each PSR. The remaining 10% are roughly equally split between 3, 2, and 1 probes for each probe set.

While junction probes provide additional information about the possible splice variants present, they do require a priori knowledge of those variants. Given that the best enumeration of splice variants to date is substantially incomplete, in particular on the whole-genome scale, the current exon arrays focus on global detection and quantification of transcribed segments which, provides a more exhaustive coverage of which regions of a gene are expressed. For a more focused custom array design, focusing on a subset of very well characterized genes that have more comprehensive information on the transcript structure and precise junction sequences, including such junction probes, in addition to all of the exon based probes, may be a good design strategy for more detailed expression analysis on these loci.

The majority of the splicing changes occur because of alternative exon usage or exon skipping, which will be captured with this type of array design. Fine modifications such as alternative 5' or 3' splice site usage with shifts of less than 25 bases will not be captured by this design.

A variety of controls are included on the Exon Array, designed specifically to facilitate the application of genome-wide exon-level expression profiling. These controls include:

• Intron controls: for approximately 100 genes with relatively constitutive expression, both exon-based and intron-based probe sets were tiled. The intron/exon normalization control probe sets can be used to monitor contamination from genomic DNA, hnRNA, as well as to provide a baseline for experiment quality control.
• Hybridization controls: bioB, vbioC, bioD and cre
• Poly-A RNA controls: lys, dap, phe, and thr

Yes, all probe and target sequences are available through the NetAffx Analysis Center.

• Information about the annotation types supporting each probe set
• Genome coordinates for all PSRs, probe sets, and probes
• Various quality information about the probe and probe sets (i.e., # overlapping probes in a probe set)
• Some biological annotation (i.e., gene names)

Approximately 190 unprocessed human microRNA sequences from the Sanger MicroRNA Registry are represented on this array. Although the probe sets are present, the current WT Sense Target Labeling Assay has not been tested or optimized to efficiently label the very small RNA molecules. Therefore, the utility of the system to measure microRNA expression is uncertain at this moment.

Based on mitomap, approximately 87 exons from the mitochondrial transcriptome are represented on this array.

The majority of the HG-U133 Plus 2.0 Array consensus sequences are well covered in the new design with approximately 75% of them containing over 8 probes on the Exon Array. The remaining 25% have less overlap with the new exon probes, and are mostly attributed to EST-based HG-U133 Array probe sets that do not contain an annotated coding region. As a result, in many such cases, the entire HG-U133 Array EST-based consensus sequence is designated as one PSR, with around 4 probes selected for that entire region.

The GeneChip™ Exon Arrays represent a new array design philosophy for the most comprehensive and informative coverage with the exon as the basic unit of expression analysis. Such an inclusive design enables discovery of new and novel splicing events not previously observed experimentally. Some of the key differences are summarized below using the human arrays as an example; refer to the "GeneChip™ Exon Array Design" Technical Note and the "Design and Performance of the GeneChip™ Human Genome U133 Plus 2.0 and Human Genome U133A 2.0 Array" Technical Note for more details.

Unfortunately, you cannot use the WT PLUS Fragmentation and Labeling kit with the SensationPlus™ Amplifcation kit. The SensationPlus™ Amp kit has reagents to go from total RNA to aRNA. The SensationPlus™ WT labeling kit has the reagents to go from aRNA to fragmented and labeled ds cDNA. The WT PLUS kit would not have the reagents to go from the aRNA to the ds cDNA portion of the protocol. Therefore the WT SensationPlus™ Labeling kit is required. Please note, this product will be discontinued. The alternative is the GeneChip™ WT Pico or GeneChip™ WT assay.

SensationPlus™ assay uses high quantity of primers to drive the conversion of FFPE RNA into productive hybridization targets and these primers cannot be completely removed by magnetic beads purification. Together with the RNA input being fragmented, the amplified nucleic acid generated by the SensationPlus™ kit are every small in size and cannot be resolved well via Bioanalyzer instrument. This is the main reason for not recommending using Bioanalyzer traces to determine the quality of SensationPlus™ kit–amplified targets for array hybridization. Please note, this product will be discontinued. The alternative is the GeneChip™ WT Pico or GeneChip™ WT assay.

No. These are completely different kits. The chemistry works a little differently in that the 3' IVT Labeling Kit for FFPE priming is done using both poly-dT and random primers. The end product is also different. It is cDNA rather than aRNA being hybridized. Please note, this product will be discontinued. The alternative is the GeneChip™ WT Pico or GeneChip™ WT assay.