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General

While mRNA should yield good results, we have not validated the product for use outside of total RNA. If you wanted to use mRNA, some optimization will have to done to determine the correct amount of input material.

We don’t recommend glycogen as a carrier since it is often contaminated with nucleic acids, sometimes has RNases, and can interfere with enzymes.

The protocol can be interrupted after:

  • IVT reaction - RNA samples can be stored overnight at -20°C
  • cRNA purification - Purified cRNA samples can be stored overnight at -20°C or at -80°C for longer - periods.
  • RNA hydrolysis after second cycle ss-cDNA synthesis

Hydrolysed ss-cDNA can be stored overnight at -20°C Purification of second cycle ss-cDNA

Purified ss-cDNA can be stored overnight at -20°C. For long-term storage we recommend that you stop here.

  • Fragmentation and labeling second cycle ss-cDNA - The fragmented and labeled ss-cDNA can be stored overnight at -20°C

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 assays generate DNA as hybridization targets, which allow for the generation of strong signals of target transcripts even in the presence of globins.

Axiom™ arrays formerly required a total of 200ng of gDNA per sample, with the exception of 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 100ng. The Axiom™ Genome-Wide Pan- African Array Set still requires a total of 300ng, or 100ng per array. Diploid plants and animals require 150ng per array and polyploid plants and animals require 200ng per array. For Axiom™ Microbiome Arrays, a total of 50ng of gDNA or 17.5 μL of cDNA reaction + 2.5 μL reduced TE buffer starting material is required per array. Please refer to the Axiom™ 2.0 gDNA sample preparation QRC for more details. Starting DNA must be double-stranded for the purpose of 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. 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. 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.

Human fecal samples and cDNA samples made from purified RNA have been validated.

DNA was extracted from fecal samples using the PowerSoil™ DNA Isolation Kit from MoBio (Cat. No. 12888-50 or 12888-100).

Blood is the only validated sample type on the assay. If you are interested in running other sample types (saliva, buccal) please contact technical support.

GeneChip™ Clariom™ S assays

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

Depending on input, sample type, and platform, you have several options:

  • Cartridge: Pico Assays for 100 pg–50 ng of total RNA isolated from whole blood, cultured cells, and fresh/fresh frozen or FFPE tissues.
  • Cartridge Assays: 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 assays.

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

Hybridization, Wash and Stain

Controls such as Oligo B2 and the 20X Eukaryotic Hybridization Controls are not included and need to be purchased separately in the GeneChip™ Hybridization Control Kit (Cat. No. 900457).

If you plan to use the stain-dispensing script, please contact your local field support specialist to have it installed or email our team at techsupport@thermofisher.com.

No. The new stain-dispensing script was designed to increase productivity and reduce the chance of error during pipetting of the stains. However, some may feel more comfortable dispensing stains by hand. For manual and automated stain-dispensing procedures, please consult the latest version of the GeneChip™ Expression Analysis Technical Manual for HT Array Plates Using the GeneChip™ Array Station (login required).

The formulation of the hybridization and pre-hybridization buffers are different; therefore, these components cannot be substituted. However, if you accidentally lose volume from one of the other five components in the HT HWS Kit (i.e., HT Stain Cocktail 1 & 3, HT Stain Cocktail 2, HT Array Holding Buffer, HT Wash Buffer A, or HT Wash Buffer B), you can supplement with the analogous components from the cartridge HWS Kit.