Search Thermo Fisher Scientific
Search Thermo Fisher Scientific
Optimize your experiments to get the best results. We’ve compiled a detailed knowledge base of the top tips and tricks to meet your research needs.
View the relevant questions below:
Having problems with your experiment? Visit our
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's 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.
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.
The following stopping points have been validated for the WT Pico assay.
- After the 14 to 16 hr IVT reaction, the cRNA samples can be stored overnight at -20 degrees C.
- After cRNA purification, the samples can be stored overnight at -20 degrees C. For long-term storage, store samples at -80 degrees C and keep the number of freeze-thaw cycles to 3 or less to ensure cRNA integrity.
- Hydrolyzed ss-cDNA samples can be stored overnight at -20 degrees C.
- Purified ss-cDNA samples can be stored overnight at -20 degrees C. For long-term storage at -20 degrees C, we recommend storing the samples as ss-cDNA and not to proceed to the fragmentation and labeling reaction.
- Fragmented and labeled ss-cDNA samples can be stored overnight at -20 degrees C. For long-term storage, we recommend storing the samples as unfragmented and unlabeled ss-cDNA.
Additional information regarding the assay workflow can be found in the GeneChip WT Pico Reagent Kit User Guide.
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.
We guarantee up to three freeze/thaws.
Axiom arrays formerly required a total of 200ng of gDNA per sample, except for the Axiom Genome-Wide Pan-African Array set, which requires a total of 300ng of gDNA per sample (100ng per array in the set).As of 2016, there are new guidelines for sample input.
All human Axiom arrays (except the Axiom Genome-Wide Pan-African Array Set) require a total of 100 ng.
The Axiom Genome-Wide Pan-African Array Set requires a total of 300 ng or 100 ng per array.
Diploid plants and animals require 150 ng per array and polyploid plants and animals require 200 ng per array.
For Axiom Microbiome Arrays, a total of 50 ng of gDNA or 17.5 uL of cDNA reaction + 2.5uL reduced TE buffer starting material is required per array.
Please refer to the Axiom 2.0 gDNA sample preparation QRC for more details. Refer to pages 12-13 of the user guide.
Quality requirements:
Starting gDNA must be double-stranded for accurate concentration determination.
gDNA must be of high purity.
DNA should be free of DNA polymerase inhibitors. Examples of inhibitors include high concentrations of heme (from blood) and high concentrations of chelating agents (i.e., EDTA).
The gDNA extraction/ purification method should render DNA that is generally salt-free because high concentrations of particular salts can also inhibit enzyme reactions.
DNA purity is indicated by OD260/OD280 and OD260/ OD230 ratios. The OD260/OD280 ratio should be between 1.8 and 2.0 and the OD260/OD230 ratio should be greater than 1.5.
DNA must not be degraded.
The approximate average size of gDNA may be assessed on a 1% agarose gel using an appropriate size standard control.
Approximately 90% of the DNA must be greater than 10 Kb in size. Control DNA can be run on the same gel for side-by-side comparison.
We recommend that DNA samples that do not meet these criteria be cleaned up as described under Genomic DNA Cleanup in the Axiom user guide.( refer to pages 15-17 in the user guide).
gDNA extracted from stool samples has been tested for use with the Axiom Microbiome Array. DNA derived from FFPE (formalin-fixed paraffin-embedded) blocks should not be used with this assay.
Any extraction and purification kit that meets the starting gDNA requirements is acceptable. However, methods that require boiling or strong denaturants should not be used, as the DNA will be rendered single-stranded.
50 ng of gDNA (20 µL at 2.5 ng/µL) is optimal. The gDNA must also be double-stranded, of high purity (OD260/OD280 ratio of 1.8-2.0 and OD260/OD230 ratio greater than 1.5), and not degraded (90% of gDNA greater than 10 kb).
The Clariom S array is a 400 format array. The correct script is FS450_0007.
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.
No, globin reduction is not necessary for the Clariom S assay. The Clariom S assay is designed to preserve sample integrity and reduce data variability without requiring a globin or rRNA removal step. This means that you can perform the Clariom S assay without the need to perform globin reduction on your samples.
Clariom™ D Assay scan time is about 33 min.
Clariom™ S scan time is about 5 min.
The GeneChip HT hybridization, Wash Stain Kit (Cat. No. 901219) is part of an older kit that is discontinued and no longer available.
The kit consisted of:
- HT Pre-Hyb mix, HT 1.2X Hyb mix, HT Stain Cocktail 1& 3, HT Stain Cocktail 2, HT Wash Buffers A, HT Wash Buffer B, and HT Array Holding Buffer.
The GeneChip HWS kit (Cat. No. 900720) is a current product that can be ordered.
This kit consists of:
- Pre-Hyb module, 2X Hyb mix, DMSO, Nuclease Free Water, Stain Module (Stain Cocktail 1 and Stain Cocktail 2) Wash Buffers, Wash Buffer B, and Array Holding Buffer.
The HT kit was formulated for high throughput usage. Therefore, the formulations differ. The Wash buffers A and B are similar, but they have slight variations in terms of additives.
The Hyb mixes have different concentrations. The stain cocktails have different stain mixtures (Stain 1 & Stain 3 vs Stain 1).
Therefore, we do not recommend swapping any of these reagents.
Only the Array Holding Buffer is the same and could be swapped.
The exact components and concentrations of the reagents are proprietary information and thus cannot be disclosed.
For Research Use Only. Not for use in diagnostic procedures.