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Frequently asked questions

  1. What kind of samples can we submit?
  2. What method should be employed to isolate my RNAs?
  3. What is the procedure if the cRNA yield is insufficient or if the cRNA quality is bad?
  4. How much RNA is necessary for a project?
  5. How many samples should be submitted?
  6. What is the RIN?
  7. How can I interpret a Bioanalyzer profile?
  8. What is the turnaround time?
  9. How to access my data?
  10. Who can access my data?
  11. How long will my data be available?
  12. How should I proceed once I download my data?
  13. Do I have to cite the Innovation Centre if I publish my research?
  14. What is the difference between the three expression profiling technologies offered?
  15. Is it recommended to compare data obtained at different times?
  16. Is it possible to compare data obtained by different technologies?
  17. Can I have my data analyzed at the Innovation Centre?
  18. Should I include sample replicates in my project?
  19. Is it mandatory to send samples on dry ice?
  20. Can I have my samples sent back to me?
  21. Why do the sample names change once they are in Nanuq?
  22. Which tests are included in the RNA QCs?
Answers
  1. What kind of samples can we submit?
  2. Only total RNA samples are accepted.

  3. What method should be employed to isolate my RNAs?
  4. We get excellent quality results with RNA samples isolated using commercial kits such as Qiagen. If your extraction protocol uses Trizol, a cleanup using Qiagen column should be done. This cleanup could be performed by the expression team if the user doesn't want to.

  5. What is the procedure if the cRNA yield is insufficient or if the cRNA quality is bad?
  6. An in-house control sample is always run alongside the users' samples. A problematic sample will be redone free of charge if our in-house control and the user samples present the same problem. However, if nothing is wrong with the control sample, the user will be asked to decide if:

    • The Innovation Centre should go ahead and hybridize all the cRNAs "as-is", knowing that the poor quality of the cRNAs sample could affect the final results. If the cRNA yield is low, all cRNAs can be normalized according to the low-yield sample. In this case, the Innovation Centre cannot guarantee a good hybridization outcome.
    • The Innovation Centre should redo the protocol with the problematic sample. If the outcome is the same, the user will have to assume the cost of the redo. There is no charge for the redo if the new cRNA is good.
    • A replacement sample should be sent.
    • The problematic sample should be dropped from the study.
  7. How much RNA is necessary for a project?
  8. The total RNA requirements are different for each technology:

    Agilent Affymetrix Illumina
    Minimum Quantity 8-samples arrays: 25ng
    4-samples arrays: 50ng
    3'arrays: 250ng
    ST arrays: 100ng
    250ng
    Minimum Volume 1.5 µl 3 µl 11 µl

    3 µl of total RNA are needed for the Quality Control tests and are not included in the volumes indicated in the above table. Moreover, it is better to submit enough material to run the experiment twice in case of a technical problem.

  9. How many samples should be submitted?
  10. The number of samples submitted depends on the technology platform and array type chosen. For example, only multiples of 12 are accepted for Illumina Human HT-12 arrays. We accept a minimum number of samples for each technology:

    Agilent Affymetrix Illumina
    Nombre minimum d'échantillons 8 6 Human HT12 et Mouse WG-6 : 12
    Mouse Ref-8: 8
  11. What is the RIN?
  12. RIN stands for "RNA Integrity Number". The RIN score is calculated by the Bioanalyzer software to determine RNA and DNA integrity. It ranges between 1 and 10; where 10 is the highest score. We do not recommend using samples with an RIN below 7.

  13. How can I interpret a Bioanalyzer profile?
  14. The Bioanalyzer profile of total RNA looks at the 18S and 28S ribosomal RNA peaks where the 28S peak should be higher. The aspect of the baseline flanking the 18S and 28S peaks is also important. It should be as close to 0 as possible and should not present any other peaks. Here are some examples of different quality total RNAs:

  15. What is the turnaround time?
  16. The turnaround time depends on the workload of the platform. Once the required paperwork is completed and we receive the samples, the Quality Control results are made available within 5 working days. If everything goes well, the final results are generally communicated within 10 working days after the reception of your arrays.

  17. How to access my data?
  18. Most of the time, final results are available through Nanuq. In your project page you need to click on "Request experimental data". Once the data are available, an automatic e-mail will be sent. You will have to come back to your project page and click on "Collect experimental data", choose the desired data and download them. If it is not possible to upload the data into Nanuq, they will be made available through a secure ftp site.

  19. Who can access my data?
  20. Only people mentioned on the initial Request Form have access to the data through Nanuq.

  21. How long will my data be available?
  22. Once you click on "Request Experimental Data", the data will be available for 15 days. After 15 days, you will have to request the data again.

    On the secure ftp site, the data will be available for only 10 days.

  23. How should I proceed once I download my data?
  24. The Innovation Centre suggest some analysis softwares for your expression data.

    - MeV (Clustering, standard differential analysis on normalysed data, etc.)
    - Bioconductor (More advanced)
    - Array Analysis
    - Microarray
    - GenomeStudio and "Gene Expression" module (not free)
    - GeneSpring (not free)

  25. Do I have to cite the Innovation Centre if I publish my research?
  26. Yes. Any collaborative work or service performed at the McGill University and Génome Québec Innovation Centre must be acknowledged and communicated to the Innovation Centre. These acknowledgements may take the form of co-authorships of platform personnel on peer-reviewed papers in which the affiliation should be clearly identified, or, the contribution of platform personnel and the Innovation Centre may simply be included in the acknowledgement section of the publications. In the case of oral communications (ex. PowerPoint presentations during scientific meetings), the use of the name Génome Québec and/or its logo is sufficient. An official reprint of each publication should then be forwarded to the Innovation Centre.

  27. What is the difference between the three expression profiling technologies offered?
  28. Agilent Affymetrix Illumina
    Species Several organisms
    Custom arrays available
    3' arrays: several species ST arrays: human, mouse, rat, CHO Mouse and human
    Number of samples Multiples of 4 or 8, depending on the array Minimum of 6 samples, no other restriction Multiples of 6, 8 or 12, depending on the array
    Quantity of starting material 8-samples arrays: 25ng
    4-samples arrays: 50ng
    3' arrays: 250ng
    ST arrays: 100ng
    250ng
    Length of probes 60nt 25nt 50nt
    Copies of each probe 1 probe per gene 3' arrays:
      - 11 copies
    Exon arrays:
      - 4 copies per exon
      - 40 per gene
    Gene ST arrays:
      - 26 copies
    6 et 8-samples:
      - 30 copies
    12-samples arrays:
      - 15 copies
    Isomers detected No 3' arrays: No
    ST arrays: Yes
    Yes
    Probes binding sites 3' 3' arrays: 3'
    ST arrays: different sites throughout the gene
    3'
  29. Is it recommended to compare data obtained at different times?
  30. Several data analysis tools allow such analyses and make it relatively simple. However, processing samples hybridized at different times introduces batch effects which make the analysis less sensitive by increasing the number of false-positives. On the other hand, the analysis specificity is higher because of the decrease of false-negatives.

  31. Is it possible to compare data obtained by different technologies?
  32. It is possible, but it is not recommended. In such comparison, a correspondence between the probes used by each technology must be made. The length, specificity and mapping of the probes being different for each technology make such a comparison very difficult.

  33. Can I have my data analyzed at the Innovation Centre?
  34. Yes, the Innovation Centre offers bioinformatics services which can help you choose the appropriate analysis tool or analyze your data for you.

  35. Should I include sample replicates in my project?
  36. In a gene expression project, the effect of different conditions on the gene expression has to be determined. Therefore, we suggest you submit at least three replicates. The more replicates you have, the more the bioinformatics analysis will be representative and statistically significant. There are two types of replicates: technical and biological. We recommend biological replicates to be used over technical replicates.

  37. Is it mandatory to send samples on dry ice?
  38. Yes. RNA samples are very sensitive to degradation. Therefore, there is a high possibility that samples received unfrozen will be degraded. The Innovation Centre will inform the users immediately when it receives samples with no dry ice. It will then be recommanded to send us replacement samples with more dry ice. We will not run unfrozen RNA samples on the Bioanalyzer unless we are requested to do so by the user.

  39. Can I have my samples sent back to me?
  40. No. The Innovation Centre policy is not to send back samples. Therefore, it is better to send us aliquots. Should the user need the samples to be sent back, an arrangement between the principal investigator and the platform manager must be made and shipping fees will apply.

  41. Why do the sample names change once they are in Nanuq?
  42. We rename all samples received in the Innovation Centre to make sure every sample has a unique name in our database (Nanuq).

  43. Which tests are included in the RNA QCs?
  44. Firstly, we measure the concentration of each RNA sample using a spectrophotometric method (Nanodrop 1000). Secondly, we calculate A260/A230 and A260/A280 ratios which indicate the presence of contaminants that could affect subsequent enzymatic reactions.

    Furthermore, we verify the RNA integrity (RIN) using the Agilent's Bioanalyzer.