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View additional product information for GeneChip™ HT WT PLUS Reagent Kit - FAQs (902414)
15 product FAQs found
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.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
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
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.