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Biopolymers Facility at Harvard Medical School
Due to the complex nature of Affymetrix Gene Chip Technology, care must be
taken during sample and chip preparation in order to maintain quality data. Below are a few of
the most frequent questions, which have been asked by our users along with their answers.
Many of these answers can be found in the Affymetrix GeneChip Expression Analysis Technical
Manual located below. If you have any comments or additional questions you'd like to see included
in this list, please feel free to email our Affymetrix technical staff directly at
affymetrix@genome.med.harvard.edu.
| What is a probe set? What do the different suffixes attached to a probe set name mean? |
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According to Affymetrix.com:
A probe set is a collection of probes designed to interrogate a given sequence.
A probe set name is used to refer to a probe set, which looks like the following:
12345_at or 12345_a_at or 12345_s_at or 12345_x_at
The last three characters (_at) identify the probe set strand. Probe sets
that are designed to detect the anti-sense strand of the gene of interest are
annotated with "_at".
There are different types of probe sets that can result from the probe selection
process. Most probe sets have an extension of an underscore and a letter to designate
the probe set type, except for unique probe sets. These different probe set types
are shown in the example above between the underscores (The first example does not have a probe set).
Probes in a gene family probe set (_a set) all cross-hybridize to the same set of
sequences that belong to the same gene family (i.e. having same name in the
"geneCluster" column). This probe set type is only created if the "geneCluster"
column is included in the Instruction File and contains information.
Probes in a unique probe set do not cross-hybridize to any other sequences in
the design (including any additional pruning sequences provided).
Probes in an identical probe set (_s set) all cross-hybridize to the same set
of sequences that are used for the design (including any additional pruning sequences
if provided). These sequences are not defined as from the same gene family for one
the following reasons: the values in the "geneCluster" column are different, or the
gene family information is not provided.
Probes in a mixed probe set (_x set) contain at least one probe that
cross-hybridizes with other sequence(s) used for the design. Cross-hybridizing probes
have a cross-hybridization penalty applied to their raw probe scores, and thus,
favoring unique probes of the same quality over cross-hybridizing probes.
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| How much labeled, fragmented cRNA must I send and at what concentration? |
| The amount of labeled, fragmented cRNA varies depending upon the type of array you are using. Test chips are MiniArrays, and most other arrays are Standard Arrays (see your chip packaging insert for this information). The final concentration in all cases should be 0.05µg/µL Micro/Mini Array: 5µg in no greater than 40µL Midi Array: 10µg in no greater than 80µL Standard Array: 15µg in no greater than 100µL While we currently do not require you to send us copies of gel photos to determine the quality and quantity of your sample, we strongly suggest that you follow the recommendations in the manual to confirm both sample quality and quantity. |
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| What is a test chip? |
| The GeneChip Test3 Array contains a subset of human housekeeping/maintenance genes shown to be expressed early on in fetal development and throughout adulthood (Physiol Genomics 2: 143-147, 2000). A smaller subset of mouse maintenance genes are also represented on the array. This set of genes, thought to be constitutively expressed to maintain cellular function, may serve as useful internal controls. The probe sequences are identical to those on the commercially available GeneChip¨ Human Genome U95 Set and GeneChip¨ Murine Genome U74 Set. |
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| Why should I run a test chip? |
| The GeneChip Test3 Array provides a convenient and accurate means of determining the quality of labeled target prior to analysis on GeneChip¨ expression arrays, allowing you to proceed with your gene expression experiments with confidence. Employing the Test3 Array as part of your gene expression experiments will save time and lead to more consistent results. The Test3 Array contains probes representing a subset of characterized genes from various organisms, including mammals, plants, and eubacteria. For each eukaryotic organism represented, probes derived from 5', middle, and 3' portions of the gene are represented. Through measurements of signals from these three probe sets, researchers can identify degraded samples containing insufficient target that would result in poor expression array results. |
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| Why won't you guarantee GeneChip results if I don't run a test chip? |
| If Test array results fall within acceptable limits, there is a high probability that the GeneChip results will be successful. If the test chip results are poor, then it is unlikely the GeneChip results will be any better. GeneChips cost in excess of $250 each whereas a test chip is only $95. Users with a great deal of experience using the Affy GeneChip system and a historical record of positive results, may wish to waive using a test chip. If you choose to do so, we still cannot guarantee successful results on the GeneChip. |
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| What exactly do you guarantee if I run a test chip? |
| If test array results fall within acceptable limits, we assume that the GeneChip results will be similar. If the test array results are good and the GeneChip results are poor we will assume that there was either operator error or a defective GeneChip. We will investigate and once we have isolated the cause, we will replace the GeneChip and repeat the hybridization and scanning procedure at no cost to you. If you do not run a Test Chip and your experiment fails, it does not mean that we will not attempt to figure out why but if an internal technical error or defective chip is not obviously found to be the cause, then we will not replace the GeneChip or repeat the experiment without a fee. |
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| Where do I hand deliver my sample to? |
| You should bring your sample to 77 Ave. Louis Pasteur, Boston, MA. We are located in room 0088 of the New Research Building. You should NEVER leave chips or samples in the facility without a staff member checking them in for you. We will NOT be responsible for lost or misplaced samples if this procedure is not followed. |
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| What if I am too far from the BPF and must ship my sample to the facility? |
| We recommend shipping the chip in a padded envelope. The cRNA sample should be in a gasketed tube, wrapped in parafilm and frozen on dry ice to avoid any possible leakage and sample degradation. You should utilize a "next morning" shipping service or "same day" cab or other delivery service to help minimize the possibility of sample degradation. Include with your chip and with your sample, the printed requisition sheet, checked and initialed by you. You will receive an email confirmation once your sample has been received. We currently do not sell GeneChips through our facility but may do so in the near future. |
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| What kits and reagents are recommended for extracting RNA from eukaryotic cells or tissues? |
| Total RNA Isolation TRIzol Reagent, Invitrogen Life Technologies, Part Number 15596-018 RNeasy Mini Kit, QIAGEN, Part Number 74104 Poly (A)+ mRNA Isolation Oligotex Direct mRNA Kit (isolation of mRNA from whole cells), QIAGEN, Part Number 72012, 72022 or 72041 Oligotex mRNA Kit (isolation of mRNA from total RNA), QIAGEN, Part Number 70022, 70042, or 70061 Qiashredder, QIAGEN, Part Number 79654 (Required only for use with QIAGEN Oligotex Direct Kit) DEPC-Treated Water, Ambion, Part Number 9920 |
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| What kits and reagents are recommended for cDNA synthesis? |
| SuperScript II, Invitrogen Life Technologies, Part Number 18064-014 SupScript Choice System for cDNA synthesis, Invitrogen Life Technologies, Part Number 18090-019 |
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| What kits and reagents are recommended for synthesis of Biotin-Labeled cRNA? |
| ENZO RNA Transcript Labeling Kit, Affymetrix, Part Number 18064-014 SupScript Choice System for cDNA synthesis, Invitrogen Life Technologies, Part Number 900182 |
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| What kits and reagents are recommended for InVitro Transcription cRNA cleanup and quantitation? |
| RNeasy Mini Kit (used in IVT cleanup), QIAGEN, Part Number 74103, 74104, 74106 CHROMA-SPIN-100 Columns in Swing Bucket Format, CLONTECH, Part Number K1302-1 DEPC-treated Water, Ambion, Part Number 9920 10X TBE, BioWhittaker, Part Number 16-012Y |
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| What kits and reagents are recommended for cRNA fragmentation? |
| Trizma Base, Sigma-Aldrich, Part Number T1503 Magnesium acetate (MgOAc), Sigma-Aldrich, Part Number M2545 Potassium acetate (KOAc), Sigma-Aldrich, Part Number P5708 Glacial Acetic Acid, Sigma-Aldrich, Part Number A628 |
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