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View additional product information for GeneChip™ 3' IVT PLUS Reagent Kit - FAQs (902415, 902416)
20 product FAQs found
Please refer to the Microarray Reagent Guide for Arrays and Expression Kits to match the correct reagents your array.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
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.
We recommend using total RNA as your input for the GeneChip 3' IVT PLUS Reagent Kit. This kit has not been validated for use with mRNA input.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
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.
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.
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.
Studies were conducted in-house to compare the data obtained from 1 µg vs. 5 µg of starting total RNA, and the array results were highly concordant with respect to detection sensitivity (percent Present call) and reproducibility (Signal correlation, Absolute Call concordance). However, subtle differences were observed. Therefore, for best comparability of results, it is generally recommended to use the same amount of starting RNA in studies where the data are intended to be directly compared.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
The IVT Labeling Kit contains a proprietary biotinylated nucleotide analog (patent pending). This analog is incorporated in the T7 RNA polymerase-mediated IVT amplification and labeling reaction as a pseudouridine reagent to generate cRNA targets.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
A linearized plasmid DNA is included in this kit as a positive control. Using this as template, users can test the transcriptional activity of the kit components. Following the protocol outlined in the package insert, the control can be used to track yield and consistency of product size generated by the kit.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
It has been shown using electrophoresis that the length of the cRNA products does not change with increasing IVT reaction time. In addition, array results generated after varying the length of IVT incubation are highly concordant with shorter incubation times and show little or no detectable difference.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
There are two major benefits for this change in protocol. One benefit is that it provides the option for starting with as little as 1 µg of total RNA using just the standard One-Cycle Labeling protocol. The other benefit is that it incorporates a more convenient and efficient experimental workflow.
Previously, IVT was carried out with 4-5 hours of incubation on the second day of a standard One-Cycle Target Labeling protocol. This made the procedure on the second day rather hectic, since users typically performed the cRNA cleanup (~30 minutes) and fragmentation (~1 hour) after the IVT in order to hybridize their samples on the arrays overnight.
In order to make the workflow more flexible, the new GeneChip IVT Labeling Kit provides a general guideline of using an overnight incubation. However, for customers who prefer the previous protocol, they have the option to spike in additional T7 RNA Polymerase (Ambion, P/N 2085, as described in the product insert) to reduce the IVT reaction time to 4-5 hours.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.