The concentration of your RNA should be carefully determined and the accessibility of the RNA for enzymes (polymerases) should be tested by RT-PCR (use primer sets that you are familiar with in your lab). Please provide information on concentration (within the GPCF Expression Profiling Submission Form) and provide gel images of the RT-PCRs performed. We will verify your information using the Agilent 2100 Bioanalyzer and the Nanodrop ND-1000 Spectrophotometer as part of our input QC.
- How many replicates need to be hybridized per experimental setting?
- What is the best protocol to extract RNA from tissues?
- Should I provide total RNA or polyA RNA for submission to GPCF?
- What is the minimum requirement for RNA quantity?
- How should I test my RNA samples before submission?
- Can I provide amplified RNA (aRNA) for hybridization at the GPCF?
- What do I need to do when I am dropping off samples for full service?
- Is there a naming convention for RNA samples?
- General terms and conditions (AGB)
The same sample on different arrays is considered a technical replicate, but the same treatment (different RNAs) on different arrays is a biological replicate. In general, the more replicates you have, the more accurate, reliable, and believable your data will be.
It is always up to each individual researcher how many replicates they do. Make sure you involve a statistician in an early phase of your experimental design ("To consult a statistician after an experiment is finished is often merely to ask him to conduct a post mortem examination. He can perhaps say what the experiment died of." Sir Ronald A. Fisher, 1938, First Indian Statistical Congress).
Technical reproducibility of Affymetrix and Illumina microarrays is highly satisfying, and we think that technical replicates are not necessary for most applications.
Biological replicates are required for impact publications of expression profiling data, to show reproducibility of gene regulation.
We recommend at least 3 replicate samples of each experimental setting for closely related cell culture approaches. Make sure to grow and harvest the cells under the most identical conditions (e.g. keeping the time between changing culture medium and harvest constant, take care to keep cell density constantâ€¦)
In animal studies of closely related individuals (e.g. when using inbred mice with different treatments), the use of >6 animal replicates per treatment is recommended. Again all environmental factors of growth, and sacrifice should be kept identical for all individuals (e.g. daytime of sacrifice, feeding and drinking status, way of tissue removal, time and treatment of tissue after removalâ€¦)
In expression profiling studies with samples of highly heterogeneous individuals (e.g. patient studies), you should always try to get access to normal and diseased tissue of the same patient. In this way you do have the most closely related "healthy" controls. Your patient cohort should be as homogeneous as possible in respect to age, sex, disease stage, disease treatmentâ€¦ It seems appropriate to do studies involving at least >15 patients of similar type.
There is not a 'best' protocol to isolate and extract RNA from tissues, but we have found that the RNeasy Kits from Qiagen produce very good results. The protocols for various different tissues and amounts are available at the Qiagen web site.
Note: Users who are using very small amounts of tissue and/or cells can use the RNeasy Micro Kit from Qiagen.
We can only accept total RNA as this can be quality controlled upon submission.
We found that one-cycle amplification/labeling is superior to two-cycle/labeling. Therefore we request at least 500 ng total RNA (at least 50 ng/Âµl) for Affymetrix GeneChipsÂ® or 500 ng total RNA (at least 30 ng/Âµl) for Illumina SentrixÂ® microarrays. For details see the GPCF Expression Profiling Submission Form.
This currently not possible as we perform full service that is highly quality controlled at all stages. The use of pre-labeled samples is not compatible with the quality standards that we apply.
When dropping off any samples to the GPCF, the GPCF Expression Profiling Submission Form has to be submitted beforehand. Make absolutely sure that the labeling of your tubes/plates is identical to the submitted sample naming, avoid "simple" naming conventions (e.g. 1, 2, 3, or A, B, C...) as these neither uniques nor informative.
Your samples should be evaluated carefully, see above "How should I test my RNA samples before submission?" (see above).
Please bring all samples in safely sealed 1,5 ml tubes, or microtiterplates (we will not accept 0,6 ml or 0,2 ml tubes).
Note: After the GPCF has evaluated the quality of the material, results of the completed assay will be accessible to the user for download. Agilent 2100 Bioanalyzer result files are provided in pdf format.
There is no naming convention for RNA samples. However, sample names must not contain "*", " ", "?", "/", "\". Also avoid "simple" namings (e.g. 1, 2, 3... or a, b, c...) and reiteration of sample names.