What starting material do I need to send you? Can you extract the nucleic acids?
For all of our microarray work, the preferred starting material is either genomic DNA in reduced TE buffer (for SNP genotyping) or total RNA in nuclease-free water (for all expression profiling work, including microRNA, do not separate the microRNA from the total RNA. We will do that if necessary). If the customer can not extract the nucleic acids, we can extract them from your samples. Thus, we can work with almost any material that contains nucleic acids: blood, cell culture lysates, saliva, tissue, etc.
How much material do you need?
The amount of material needed is based on the type of assay to be performed including material used for QC:
For expression profiling we request:
1000 ng of total RNA for 3’-biased array processing
500 ng of total RNA for Gene Arrays
1000 ng for Exon arrays
3 µg for microRNA expression
11 µg of RNA for Prokaryotic expression profiling
For Genotyping (using the SNP array 6.0) we request 2 µg of DNA.
What are the minimum QC requirements for the nucleic acids?
We like RNA to meet the following minimums:
OD260:280 > 1.80
OD260:230 > 1.80
RIN# 8.0 (as assessed by Agilent Bioanalyzer 2100)
It may still be possible to process RNA that does not meet these minimums but that is determined on a case-by-case basis.
For DNA:
OD260:280 > 1.60
OD260:230 > 1.60
Genomic DNA mostly intact as assessed qualitatively on an agarose gel
Concentration between 50 & 100 ng/µl
What if my material does not meet the minimums?
In many cases, we can work with material that falls a below these values. In these instances, it is really a judgment call made by our lab supervisors in consultation with the customer. If we encounter sub-optimal samples we will contact the customer, discuss the relative risks and options and if approved by the customer, we will move forward.
Can you work with samples from formalin-fixed, paraffin-embedded (FFPE) samples?
In short, yes. If the customer is considering working with FFPE samples we strongly encourage them to use the Exon or Gene arrays instead of the 3’-biased arrays. The Exon and Gene arrays use a random priming approach to amplification (as opposed to a 3’-biased approach) and are therefore less sensitive to higher levels of fragmentation in the starting material. Although the assay is a little more expensive than the 3’ assay, the customer will have to reject fewer samples and thus should get more data in the long run.
What kind of data will I get back? How much bioinformatics analysis is included?
The customers get back all of the raw data files (.dat, .cel, .rpt, etc.) produced by the Affymetrix system as well as a report with both the initial sample QC showing the nature of the starting material and post-hybridization QC showing the validity of the assay. For more gene expression experiments, if requested, we will prepare an A vs. B gene list detailing the genes that are significantly up- and downregulated in the experimental group(s) when compared to the designated control group. For SNP experiments, we provide a list of genotype calls for each of the samples. More involved analyses and more complex bioinformatics can be negotiated into a project, sometimes at no additional cost or for a small additional fee. Our bioinformatics services are usually charged on an hourly basis.
What kind of process/protocols do you use?
That really depends on the nature of the samples and what is necessary to achieve the desired analysis. We tend to primarily use the Affymetrix assay chemistries, but after our complimentary experimental design session with our customers, we may deem that Genisphere, NuGEN, or other manufacturers’ assays maybe more appropriate for the project.
Do you offer any microarray platform other than Affymetrix?
Not at this time, although other platform technologies are always being evaluated and may be incorporated in the future as deemed appropriate.
Are you Affymetrix? OR Are you affiliated with Affymetrix?
No. We are one of a list of authorized service providers that offer Affymetrix microarrays but we are an independent company.
What species can be analyzed with Affymetrix chips? Can I get a custom chip made for my organism of interest?
The list of species for which Affymetrix makes microarrays is available on their website. There are over 30 species for which RNA expression profiling chips are available. SNP genotyping is currently available only on human and canine chips. The microRNA profiling chip contains probes to microRNA from more than 70 different species. We are happy to process custom chips but we do not synthesize or sell these chips ourselves. If requested, we will work with customers in designing and arranging manufacture of the chips. Keep in mind that there are certain restrictions on how many or few chips are available in custom formats, since the customer must commit to a production run of the custom chips and therefore must purchase whole wafers (instead of individual chips).
What other services does PBR offer?
Besides microarray processing we perform nucleic acid extractions for customers from a variety of samples. In addition we can do qPCR and RT-qPCR analysis. Finally, we have a staff of bioinformatics people that can assist in data mining and the analysis of customer datasets.
How do you validate findings?
Validation is a tricky subject and the method used must be chosen in the context of the question. While qPCR and RT-qPCR have been and continue to be used, they are not always the most appropriate. Customers should consider proteomics, histochemistry or other approaches as appropriate to their experimental system and the question being asked. Our staff is willing to consult with the customer in determining the best approach and if possible will even work to identify other providers if Precision Biomarker Resources is unable to do the validations in house.
Can you work with laser-capture microdissection (LCM) samples?
Precision Biomarker Resources staff has experience with LCM samples having successfully used them in expression profiling experiments in the past. There are several protocols available for extremely limited sample amounts and the choice of protocol is influenced by the nature and amount of sample to be processed, the type of microarray to be used and other factors. Please call and speak with someone from our technical staff before beginning any LCM work in order to optimize customer chances of a successful experiment.
Do you recommend technical replicates?
No. We have found the major source of experimental variability to be in the biology of the system and not the technical end. The manufacturing of microarrays and the processing of samples has achieved a very high degree of reproducibility. While not 100%, it is far overshadowed by the variability inherent in any biologic system. For this reason, we recommend that where the money for replication is available that it be spent on biologic replication (i.e. replicate cultures or multiple animals) and not technical replications (i.e. processing the same sample more than once).
Why do you recommend a minimum of triplicate determinations for expression profiling work?
Anything less than triplicates makes statistical evaluation of the data impossible. Singlet observations are anecdotal at best. Even duplicates do not give you the power to do solid analyses. (If samples do not match, how do you know which is the outlier?) Triplicates are the minimum valid experimental design for statistical analysis and, as with any other testing, the more replication you can do, the more powerful your statistics will be.
