University of Pittsburgh Cancer Institute (UPCI)

CCSG Acknowledgement

Required CCSG Acknowledgement

The NCI requires that publications acknowledge the UPCI CCSG support, and they are tracking compliance. If a UPCI CCSG-supported Shared Resource provided data used in your publication, please include the following statement in the acknowledgment section of your publication(s):

"This project used the UPCI [insert name(s) of shared resource(s)] that [is/are] supported in part by award P30CA047904."

Shared Resource Directors: Please make sure to include this statement on all of your order forms, contracts, etc. as a reminder to your users to acknowledge the UPCI CCSG support.

RNA Expression: Exiqon

Click here to order an Affymetrix GeneChip expression assay.

The CBF utilizes the Exiqon miRCURY LNA™ Power Labeling assay to generate hybridization targets from total RNA containing miRNA.

The miRCURY LNA™ microRNA Power Labeling assay provides a fast and simple method for fluorescent labeling of total RNA samples from animals, plants and viruses. All small RNAs are uniformly labeled and the labeled RNA molecules may be hybridized directly to the miRCURY LNA™ arrays, without additional microRNA enrichments or other time-consuming handling steps.

The miRCURY LNA™ microRNA Power Labeling assays follow a simple and fast two-step protocol. First, 5'-phosphates are removed from the microRNA termini using Calf Intestinal Alkaline Phosphatase (CIP). Second, a fluorescent label is attached enzymatically to the 3'-end of the microRNAs in the total RNA sample. This is followed by an enzyme inactivation step after which the sample is ready for hybridization.

Investigators may choose to have the CBF extract and prepare RNA for them directly from frozen tissues or cell samples, or they can choose to submit investigator-prepared RNA. If you choose to extract and submit your own RNA, please note: You must prepare total RNA containing miRNA; do not prepare samples enriched for miRNA!

High quality RNA is critical to the success of a microarray expression assay, so all samples generated within the CBF and investigator-prepared samples will be assessed for quality via the Nanodrop spectrophotometer and the Agilent Bioanalyzer. Investigators will be notified of all samples that do not meet Quality Control criteria for quantity and quality. Please see Sample Requirements below for more information.

Exiqon miRNA arrays are available for human, mouse, and rat in a single array format as well as for many other eukaryotic and prokaryotic model organisms.

miRCURY LNA™ microRNA Array, 5th gen - hsa, mmu & rno
The 5th generation arrays contains more than 1891 capture probes, covering all human, mouse and rat microRNAs annotated in miRBase 14.0, as well as all viral microRNAs related to these species. In addition, this array contains capture probes for 385 new miRPlus™ human microRNAs. These are proprietary microRNAs not found in miRBase.

miRCURY LNA™ microRNA Array, v. 11.0 - Other Species
This array contains close to 2500 capture probes from species other than human, mouse and rat. It has a unique coverage of 4168 mature microRNAs from 85 species, and can be used for microRNA profiling from vertebrates, invertebrates, plants and viruses and for cross-profiling between species.

Sample Requirements

Expression Array Type Target Prep Method *^Minimum Amount of Total RNA Containing miRNA Required Minimum Concentration
5th Generation HAS, MMU, RNO Hy3 or Hy5 Power Labeling Kit 500ng 250ng/ul
microRNA Array, v. 11.0 -Other Species Hy3 or Hy5 Power Labeling Kit 500ng 250ng/ul
*Quality, Quantity, and Concentration subject to verification by the CBF
^ These are the minimal amounts specified in the respective protocols. We recommend you provide us with excess in order to run QC analysis and to repeat assays if necessary

High quality array data depends upon high quality samples!

RNA: Samples intended for the Exiqon miRNA assay must be total RNA containing miRNA; do not prepare samples enriched for miRNA! The CBF recommends using the Qiagen miRNeasy kit for extraction.

Prior to target synthesis, the CBF will evaluate all submitted RNA samples for quantity and quality (amount of degradation). RNA will be quantified by UV absorbance using a Nanodrop spectrophotometer and validated to be non-degraded via the Agilent Bioanalyzer. A260:A280 ratios should be 1.8-2.0 and the RIN should be >7.0. Submit RNA in nuclease-free water.

Tissue or cells may also be submitted to the CBF for extraction prior to performing an expression array assay. For researchers who are collecting tissue on their own, we suggest a few guidelines to limit RNA degradation and ensure sample quality. All samples submitted to the lab for extraction and downstream microarray applications should be handled in one of the following ways at the time of collection:

  • Fresh tissue should be flash frozen in 2.0 mL or Nunc tubes in LN2 or dry-ice and maintained at -80°C until submitted to the CBF. Cells should be pelleted in 1.5-2.0 mL tubes, all supernatant removed, and then flash frozen in LN2 and maintained at -80°C until submitted to the CBF.
  • Tissue and cell pellets should be placed in RNAlater (Ambion, Inc) and stored at 4°C for up to one month, or
    Tissue and cell pellets should be placed in RNAlater at 4ºC overnight, then stored at -20°C indefinitely until submitted to the CBF, or
    Tissue should be placed in RNAlater at 4°C overnight, then removed from the RNAlater and stored at -80°C indefinitely until submitted to the CBF. Cell pellets should be placed in RNAlater at 4°C overnight, then stored at -80°C indefinitely until submitted to the CBF.
  • Tissue and cell pellets should be homogenized immediately in either Qiagen Buffer RLT (recommended) or Trizol (not recommended) and then stored at -80°C until submitted to the CBF.

Once samples have been extracted they will be assessed for quality, quantity, and concentration prior to beginning microarray assays.

Investigators will be notified if a sample is of insufficient quantity or quality (failed Quality Control parameters) for optimal array performance.

Samples cannot be processed until a completed Service Request Form ) is received along with a signed estimate. Click here for pricing information.

If you would like to order an array that is not listed, please contact the CGIF.

Technology and Equipment

Exiqon Products

Exiqon miRCURY LNA microRNA Arrays

View a complete list of miRNA arrays that can be run in the CBF.

Exiqon's microRNA arrays feature Tm-normalized LNA™-enhanced capture probes, designed for excellent specificity and sensitivity even for AT-rich microRNAs. In addition, they offer great reproducibility with 99% correlation between arrays and a dynamic range greater than 5 orders of magnitude.

Genepix 4000B Microarray Scanner

Genepix 4000B Microarray Scanner

The GenePix 4000B Microarray Scanner acquires data at two wavelengths simultaneously. This means drastically reduced scan times and hence greater workflow efficiency. Apart from superior speed, simultaneous scanning eliminates potential errors associated with aligning two separately scanned images, and provides real-time access to data as they are acquired.

With automated photomultiplier (PMT) balancing, the fully integrated GenePix Pro Acquisition and Analysis Software controls the GenePix 4000B Microarray Scanner and can automatically evaluate the required PMT gain values, for fast and easy optimization of signal intensity and channel balance. The GenePix 4000B Microarray Scanner then acquires data at your selected resolution between 5-100 micron, allowing the adjustment of image resolution and file size for each experiment.