GeneChip™ WT PLUS Reagent Kit, 30 Reactions - FAQs

Please refer to the Microarray Reagent Guide for Arrays and Expression Kits to match the correct reagents your array.

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Sequences of all our controls can be downloaded from the sequence file that is available on product pages on thermofisher.com.

For example, here is the link to the product page for Clariom D Assay, human: https://www.thermofisher.com/order/catalog/product/902923
Go to the "Software & Data Analysis" section and under "Support Files", click on "Sequence Files: Clariom D Array, human Probe Sequences, tabular format", which you can open in Excel.

Note: If the control sequence file is not available, please contact techsupport@thermofisher.com.

Pseudogene databases were not included in the design of expression arrays.

The first dilution of the Poly-A RNA Control can be stored at -20 degrees C for 6 weeks (with up to 4 freeze/thaws).

Labeled material can be stored for 2 weeks at -20 degrees C.

Typical shelf life for expression arrays are as follows:
- All ≥ 8 µm commercial expression cartridges are stable up to 24 months.
- All ≥ 8 µm commercial resequencing cartridges are stable up to 18 months.
- All ≥ 8 µm commercial expression plates are stable up to 18 months.

Please note that this excludes products such as Exon or Gene array as they are 5 µm cartridge and plates.

For IVT assays, the RIN should be 6 or higher. For WT assays, there are no recommendations based on assay priming.

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The protocols for the Gene 1.0 ST and Exon 1.0 ST arrays are the same until the fragmentation and labeling steps.
Depending on the input amount, please refer to the GeneChip WT Pico Reagent Kit or the GeneChip WT PLUS Reagent Kit for the protocol:
- WT Pico: 50 - 500ng of total RNA
- WT PLUS: ≥ 100pg of total RNA (approx. 10 cells)
From the hybridization step onwards, the differences in protocol are due to the format of the arrays. The Exon array is a 49 format array, whereas the Gene 1.0 ST array is a 169 format array.
For the fluidics step the following scripts should be used depending upon which array you are using:
- Gene 1.0 St array: FS450_0001
- Exon 1.0 St array: FS450_0007

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All senseRNA, cRNA, and cDNA products generated using GeneChip WT PLUS Reagent Kit and GeneChip 3' IVT PLUS Reagent Kit can be stored at -20 degrees C.

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We guarantee up to three freeze/thaw cycles for the GeneChip WT Plus Reagent Kit.

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We recommend the following assays dependent upon your respective sample size:
- WT Plus Assay for 50-500 ng of total RNA isolated from whole blood, cultured cells, and fresh/fresh frozen tissues.
- WT Pico Assay for 100 pg - 50 ng of total RNA isolated from whole blood, cultured cells, and fresh/fresh frozen or FFPE tissues.

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Extremely high cRNA yield when using a GeneChip WT PLUS Reagent Kit can be caused by gDNA contamination. Depending on the level of gDNA contamination, low array signals and low positive vs. negative AUC can occur.

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Depending on the sample type, input RNA amount, and required instruments, there are several cartridge and array plate options.
- Cartridge with GeneChip Pico Kit for 100pg–50 ng of total RNA from whole blood, cultured cells, and fresh/fresh-frozen or FFPE tissues.
- Cartridge with GeneChip WT Plus Reagent Kit for 50–500 ng of total RNa isolated from whole blood, cultured cells, and fresh/fresh-frozen tissues.
- 24 or 96-array plate with GeneChip Pico Kit for 100pg–50 ng of total RNA from whole blood, cultured cells, and fresh/fresh-frozen or FFPE tissues, for analysis on the GeneTitan MC instrument.
- 24 or 96-array plate with GeneChip WT Plus Reagent Kit for 50–500 ng of total RNA isolated from whole blood, cultured cells, and fresh/fresh-frozen tissues, for analysis on the GeneTitan MC instrument.
Additional information about the Clariom S options offered can be found in the Data Sheets: Clariom S Solutions document on the Thermo Fisher website.

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Yes, the GeneChip microarrays can use either poly(A)+ mRNA or total RNA as input material. The protocols provided in the manual describe procedures for preparing biotinylated target RNA from both purified eukaryotic poly(A)+ mRNA and total RNA samples. Good-quality mRNA has been successfully isolated from mammalian cells and tissues using specific kits, and this mRNA can be used as a template for cDNA synthesis. However, it is important to note that results obtained from samples prepared using different methods (total RNA vs poly(A)+ mRNA) may not be identical, so it is recommended to only compare samples prepared using the same sample preparation protocol.

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Yes, high amounts of ribosomal RNA (rRNA) can influence the amplification process. Ribosomal RNAs are the most abundant types of RNA present in cells, and they can compete with messenger RNAs (mRNAs), which are usually the targets that bind to the probes on the array, during the reverse transcription and amplification steps. This competition can lead to less efficient amplification of the mRNAs, which can impact the overall quality and reliability of the outcome data.
However, it's important to note that this doesn't necessarily cause "random" primer amplification.
The primers used in these steps are designed to be specific for the mRNAs depending on the assay used and also on the probes on the array, so the presence of excess rRNA shouldn't cause them to bind and amplify random sequences. But it can reduce the efficiency with which they amplify the specific sequences for which the probes are designed for.
As a best practice, depending on the nature of the input , we often recommend that you deplete rRNA from the samples before performing these steps, especially when working with total RNA samples. There are several commercially available kits and methods for rRNA depletion.

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We do not avoid globin through primer design for the GeneChip microarrays. All our assays label and/or amplify globin RNA, and the amplified globins hybridize and generate array signals.
However, our WT (whole transcript) assays generate DNA as hybridization targets, which allow for the generation of strong signals of target transcripts even in the presence of globins. This is in contrast with the IVT (in vitro transcription) assays, where the binding of the generated labeled RNA targets and the array signals are weaker, due to globin interference. Therefore, it is necessary to run a globin reduction when using the IVT assays.

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Total RNA samples should be free of genomic DNA and we recommend including a DNase treatment with the RNA purification method. Contaminating genomic DNA may be amplified along with the RNA, which can lead to inaccurate measurement of whole transcriptome expression. In addition, the contaminating genomic DNA could cause over-estimation of the RNA amount.​
We strongly recommend against the use of nucleic acid-based and those that contain glycogen carriers during RNA purification because many have been shown to produce cDNA products in first-strand synthesis reaction. Choose a purification method or commercially available kit that is appropriate for your sample amount. For limited cell numbers, choose purification methods that enable purification of total RNA preparations from small amounts.

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Control Oligo B2 (3 nM) is a pre-labeled DNA spike-in control required for hybridization to the control probes on the array utilized for grid alignment by AGCC. A failure to include Control Oligo B2 in the hybridization cocktail will result in the inability of the software to apply a grid over the scanned image, leading to the unrecoverable loss of sample data from the array. The absence of the Control Oligo B2 also voids the customer's ability to submit an array replacement request.

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The WT Plus kit or the WT Pico kit is recommended for preparing samples for use with the Gene 1.0 ST Array and the Gene 2.0 ST Array.

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Utilizing the WT assay for partially degraded samples may be an attractive strategy for profiling these samples. However, it has not been tested thus far in development; therefore, it is recommended that only high-quality total RNA samples should be used.

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The WT Plus Assay is optimized to produce targets specifically for hybridization to the Whole Transcriptome(WT) type of design. The target is in the sense orientation and the GeneChip Human Genome U133 Plus 2.0 Array is designed to be compatible with anti-sense targets. Therefore, it is not recommended to mix and match the assays and the array types.

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There are a few safe stopping points in the assay, including:

-After IVT reaction and the cRNA cleanup step in the first cycle, before proceeding to the second cycle of reverse transcription
-After reverse transcription and the single-stranded cDNA cleanup step in the second cycle, before fragmentation and labeling
-After fragmentation and labeling, before hybridization


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On a Bioanalyzer, the fragmented single-stranded DNA target should have a peak centered around 40 to 70 bases with the majority of the fragments ranging from 20 bases to 200 bases.

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Using cRNA generated from the IVT reaction at the end of the first cycle of the assay as a template, single-stranded DNA is synthesized using random primers and the dUTP + dNTP mix. The resulting single-stranded DNA (ss-DNA) containing the unnatural uracil base is then treated with Uracil DNA Glycosylase, which specifically removes the uracil residue from the ss-DNA molecules. In the same reaction, the APE 1 enzyme then cleaves the phosphodiester backbone where the base is missing, leaving a 3'-hydroxyl and a 5'-deoxyribose phosphate terminus.

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A total of 100 ng of total RNA per sample is the recommended starting quantity if following the standard protocol. A total of 50 ng can be used at a minimum.

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By titrating genomic DNA back into the total RNA samples and monitoring the deterioration of the array data, it was determined during development of the assay that a moderate amount of genomic DNA contamination will only have minimum effect on the array results. Therefore, routine RNA isolation techniques coupled with DNase treatment should yield sufficiently high-quality sample for analysis on the exon arrays.

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