FAQS

Do you have technical questions about our services? Do you want to know more about a specific analysis technique? Check out these links to find out more.

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Genotyping

What technologies and platforms do you use?

Depending on your experiment and the type of samples you have, we will recommend the most appropriate methodology and platform. Of course we are happy to accept your direction on those choices, should you have a preference.

What starting material do I need to send you? Can you extract the nucleic acids?

The preferred starting material is genomic DNA in reduced TE buffer. If you can’t extract the nucleic acids, don’t worry—we can do it for you. Just send the samples. We can work with almost any material that contains nucleic acids: blood, cell culture lysates, saliva, tissue, etc.

How much material do you need?

For genotyping with SNP array 6.0, we request 2 µg of DNA.

  • OD260:280 > 1.60
  • OD260:230 > 1.60
  • Genomic DNA "mostly intact" as assessed qualitatively on an agarose gel concentration >100 ng/µl

What if my material does not meet the minimums?

In many cases, we can work with material that falls below the above values. In these instances, it is really a judgment call made in consultation with you. Give us a call to discuss the relative risks and options.

What kind of data will I get back? How much bioinformatics analysis is included?

You get all raw data files as well as a final report that includes initial sample and post-hybridization quality control. We also 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.

Can the SNP 6.0 be used for copy number analysis?

Yes.  Using the SNP 6.0 array, our bioinformatics team can look at copy number variations.

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Expression Profiling

What technologies and platforms do you use?

Depending on your experiment and the type of samples you have, we will recommend the most appropriate methodology and platform. Of course we are happy to accept your direction on those choices, should you have a preference.

What starting material do I need to send you? Can you extract the nucleic acids?

For all of our expression microarray work, the preferred starting material is total RNA in nuclease-free water. For microRNA, do not separate the microRNA from the total RNA. If you can’t extract the nucleic acids, don’t worry—we can do it for you. Just send the samples. 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 we request—including material used for quality control—is 1-2 µg of high-quality total RNA for most eukaryotic expression arrays, and 3-5 µg of total RNA for prokaryotic expression profiling, at concentrations > 100 ng/µl.

What are the minimum quality control 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)

What if my material does not meet the minimums?

In many cases, we can work with material that falls below the above values. In these instances, it is really a judgment call made in consultation with you. Give us a call to discuss the relative risks and options.

Can you work with samples from formalin-fixed, paraffin-embedded (FFPE) samples?

Yes. If you want to work with FFPE samples, we strongly encourage you to use the Exon or Gene arrays instead of 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. You will have to reject fewer samples and should get more data in the long run.

What kind of data will I get back? How much bioinformatics analysis is included?

You get all raw data files as well as a final report that includes initial sample and post-hybridization quality control. If requested, we will prepare "A vs. B" gene lists detailing the genes that are significantly up- and down-regulated in the experimental group(s). 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.

How do you validate findings?

Validation methods must be chosen to suit your research question. While RT-qPCR is commonly used, it is not always the most appropriate. We can take you through a range of other options, including proteomics and histochemistry. We will discuss and implement the best approach in consultation with you.

Can you work with laser-capture microdissection (LCM) samples?

Yes, we have successfully used LCM samples in expression profiling experiments. There are several protocols available for extremely limited sample amounts. The choice of protocol is influenced by several factors, such as the nature and amount of sample to be processed and the type of microarray being used. Please call and speak with us before beginning any LCM work to get some advice on how to optimize results.

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qPCR

Do you design probes and primers for qPCR experiments?

Yes. Our bioinformatics team can design probes and primers targeting your genes of interest.

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Next-gen sequencing

What sequencing services does PBR offer?

PBR offers a wide range of next-gen sequencing services. RNAseq is the most requested, but we also provide other services, such as exome sequencing, target region sequencing and small RNA sequencing. Contact us to discuss additional services, such as whole genome resequencing, ChIP-seq applications, and others.

What are the sample requirements for RNAseq?

RNA should be free of protein (OD 260/280 of 1.8-2.2) and treated with DNase to remove DNA contamination. For mammalian cells/tissues, a minimum of 5ug (80-100ng/ul) total RNA should be submitted, although 10ug is preferred, to allow for quality control. For all other species, 10ug (200ng/ul) should be submitted. PBR can also extract nucleic acids for you to ensure the highest quality samples. Call us to discuss your options.

What sample quality control (QC) does PBR perform?

PBR performs intake QC on all samples as soon as they are received. Specifically, we spectrophotometrically determine sample concentration with OD260 and purity with OD260/OD280 between 1.80 and 2.20. RNA integrity is determined using an ABI2100 BioAnalyzer; we look for RIN value of 8.0 or greater with a 28S/18S ratio of at least 1.0.

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Explore data

Why do you recommend a minimum of triplicate replications?

Anything less than triplicates make statistical evaluation of 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. Replicates of three or greater allow us to use t-tests or ANOVA to determine if the differences seen are real.

Do you recommend technical replicates?

No, not usually. We have found the major source of experimental variability to be in the biology of the system. The manufacturing of microarrays, and the processing of samples, has achieved a very high degree of reproducibility. While not 100%, we recommend prioritizing biologic replication (i.e., replicate cultures or multiple animals) rather than technical replications. However, as with most things in experiment design, one size does not fit all. If you are concerned that technical replication may be needed, we encourage you to call us to discuss your specific project.

Where can I get a list of genes, SNPs or miRNA represented on my GeneChip array?

The annotation files for each chip type are on the Affymetrix website. The NetAffx section can be particularly useful. If you have trouble navigating the Affymetrix website, please give us a call and we will guide you through the chip types.

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