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.


Genomics Services: DNA Extraction and QA/QC

DNA and RNA extraction and quality control of the extracted nucleic acids are crucial for sequencing, microarray analysis, or other genomic technologies. The CBF has established standard operating procedures for extractions to minimize degradation and contamination of precious patient samples. Quality analysis is performed with the Nanodrop ND1000 spectrophotometer, the Qubit Fluorometer, and the Agilent 2100 Bioanalyzer, which has become the industry standard for RNA quality assessment.

The CBF offers the following extraction and quality analysis services:

  • DNA Extraction via Qiagen DNeasy kits
  • DNA Extraction from whole blood via Qiagen Flexigene kit
  • DNA Deparaffinization
  • RNA Extraction via Qiagen RNeasy kits
  • miRNA Extraction via Qiagen miRNeasy kits
  • RNA Extraction from FFPET samples via the Agencourt FormaPure and Ambion RecoverAll kits
  • RNA Quality Analysis via Agilent Bioanalyzer
  • Nucleic Acid Quantitation via Nanodrop ND1000
  • Nucleic Acid Quantitation via the Qubit Fluorometer

Sample Requirements

DNA ExtractionTissue samples may be requested through the UPCI Tissue and Research Pathology Services (TARPS) and cell culture samples may be requested through the CBF (contact Patricia Petrosko).

For researchers who are collecting tissue and cells 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 4°C 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.
  • FFPET samples may be submitted in one of two ways:
    • Submit (10) 10 micron scrolls in round bottom 2.0 mL microcentrifuge tubes
    • Submit (10) 10 micron blank slides for FFPET extraction. The CBF can perform macro-dissection of the FFPET slides. The researcher must provide an accompanying H&E slide with the area of interest outlined. If this service is requested, we suggest that the researcher talk with a CBF staff member prior to submitting samples to go over the protocol for identifying the area of interest.

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.

Equipment

Agilent Bioanalyzer

Agilent Bioanalyzer

The Agilent 2100 Bioanalyzer is a microfluidics-based platform for sizing, quantification and quality control of DNA, RNA, proteins and cells on a single platform. Learn more at the Agilent website.

Nanodrop SpectrophotometerNanodrop Spectrophotometer

The Nanodrop (Thermo Scientific) is a micro-Volume UV-Vis spectrophotometer for nucleic acid and protein quantitation that allows for sample volumes as small as 0.5 μL. It offers:

  • Direct, easy measurements in less than 5 seconds - just pipette & wipe. No cuvette or dilutions!
  • Full spectral output
  • Measures DNA, RNA (A260) and Protein (A280)
  • concentrations and sample purity (260/280 ratio)
  • Large concentration range (2 ng/μL - 15,000 ng/μL dsDNA) without dilutions

Learn more at the Nanodrop website.

Life Technologies Qubit Fluorometer

Qubit FluorometerThe Qubit® 2.0 Fluorometer utilizes specifically designed fluorometric technology using Molecular Probes® dyes. These fluorescent dyes emit signals ONLY when bound to specific target molecules, even in the presence of free nucleotides or degraded nucleic acids. Qubit® fluorometric quantitation provides the most specific and sensitive DNA and RNA quantitation available, even at low concentrations.

Learn more at the Life Technologies website.