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Sample Quality

Contents

Introduction

The Microarray Core Facility has purchased several instruments for analysis of sample quality including an Agilent Bioanalyzer, an Invitrogen Qubit, and a NanoDrop spectrophotometer. Appropriate quality control tests using these instruments are performed on all samples that are submitted to the facility for microarray or sequencing services. Samples must pass quality control measurements prior to proceeding with further steps in the experimental pathway. Investigators will be contacted if any samples fail at this stage of the experimental process. Alternatively, investigators can also submit experiment requests for "Sample Quality" only and select the appropriate quality control step that they would like to be performed on their sample.

Bioanalyzer RNA 6000 Nano Chip

  • The RNA Nano Chip provides a qualitative assessment of the integrity of a total RNA sample. The bioanalyzer software also provides a quantitative estimate of the sample and calculates the 28S/18S ribosomal RNA ratio. This chip has been optimized for a quantity of total RNA that ranges from 25 to 500 ng.
  • Provide a volume of 3 μl of purified total RNA in water (or TE) at a concentration between 25 ng/μl and 500 ng/μl for samples to be run on the Bioanalyzer RNA 6000 Nano Chip.
  • Do not send a sample volume/quantity in excess of the guidelines above. We are unable to provide a long term storage solution for excess samples. The unused portion of these samples is discarded after the experiment has been successfully completed.
  • A RIN (RNA integrity number) number is calculated for each total RNA sample that is run on the bioanalyzer. The RIN number provides an assessment of the quality of the RNA sample on a scale of 1 to 10. An application note that describes this assessment can be found at [1].
  • Pricing for Sample Quality Services including Bioanalyzer RNA 6000 Nano Assay

Bioanalyzer RNA 6000 Pico Chip

  • The RNA Pico Chip provides a qualitative assessment of the integrity of a total RNA sample. The bioanalyzer software also provides a quantitative estimate of the sample and calculates the 28S/18S ribosomal RNA ratio. This chip has been optimized for a quantity of total RNA that ranges from 50 to 5000 pg.
  • Provide a volume of 3 μl of purified total RNA in water (or TE) at a concentration between 50 pg/μl and 5000 pg/μl for samples to be run on the Bioanalyzer RNA 6000 Pico Chip.
  • Do not send a sample volume/quantity in excess of the guidelines above. We are unable to provide a long term storage solution for excess samples. The unused portion of these samples is discarded after the experiment has been successfully completed.
  • A RIN (RNA integrity number) number is calculated for each total RNA sample that is run on the bioanalyzer. The RIN number provides an assessment of the quality of the RNA sample on a scale of 1 to 10. An application note that describes this assessment can be found at [2].
  • Bioanalyzer data can be downloaded from GNomEx by clicking on the “Experiments” tab, selecting the experiment request of interest, clicking the download link and selecting the desired folders to download.
  • Pricing for Sample Quality Services including Bioanalyzer RNA 6000 Pico Assay

Bioanalyzer DNA 1000 Chip

  • The Bioanalyzer DNA chip is used for evaluating the size distribution of Illumina Library preparations, pcr products, and restriction digests. The size range of samples that can be resolved on the DNA 1000 chip is 25-1000 bp.
  • Provide a volume of 3 μl of purified DNA in TE at a concentration between 5 ng/μl and 50 ng/μl for samples to be run on the Bioanalyzer DNA 1000 Chip.
  • Do not send a sample volume/quantity in excess of the guidelines above. We are unable to provide a long term storage solution for excess samples. The unused portion of these samples is discarded after the experiment has been successfully completed.
  • Bioanalyzer data can be downloaded from GNomEx by clicking on the “Experiments” tab, selecting the experiment request of interest, clicking the download link and selecting the desired folders to download.
  • Pricing for Sample Quality Services including Bioanalyzer DNA 1000 Assay

Bioanalzyer DNA High Sensitivity Chip

  • The Bioanalyzer High Sensitivity DNA kit is used for evaluating the size distribution of fragmented samples to be used for chromatin immunoprecipitation. The size range of samples that can be resolved on the High Sensitivity DNA chip is 50-7000 bp.
  • Provide a volume of 3 μl of purified DNA in TE at a concentration between 5 pg/μl and 500 pg/μl for samples to be run on the Bioanalyzer High Sensitivity DNA Chip.
  • Do not send a sample volume/quantity in excess of the guidelines above. We are unable to provide a long term storage solution for excess samples. The unused portion of these samples is discarded after the experiment has been successfully completed.
  • Data can be downloaded from GNomEx by clicking on the “Experiments” tab, selecting the experiment request of interest, clicking the download link and selecting the desired folders to download.
  • Pricing for Sample Quality Services including Bioanalyzer DNA High Sensitivity Assay

Qubit PicoGreen Assay

  • The measurement of ChIP-DNA sample concentration on a NanoDrop tends to be very inaccurate due to the high percent of background measured in the A260 wavelength in dilute concentrations of nucleic acid.
  • The Qubit fluorometer (Invitrogen) uses the Quant-iT dsDNA HS Assay Kit is used to measureDNA concentration. This assay uses PicoGreen to discriminate between various nucleic acid contributors in the sample and provide an accurate measurement of the concentration of dsDNA in the sample. This assay can quantitate samples that range from 10 pg/μl to 100 ng/μl.
  • ChIP-DNA samples typically require this more-sensitive pico-green based assay to provide an accurate assessment of the DNA concentration. ChIP-DNA samples are typically observed to have a concentration that is less than 1 ng/ul.
  • A minimum of 10 μl of each sample should be provided for measurement. Ideally, only 1 μl of sample will be used for the measurement and the remainder will be returned. However, many ChIP-DNA samples are severely dilute and the assay may take as much as 5-8 μl of sample.
  • Pricing for Sample Quality Services including Qubit PicoGreen Assay

Genomic DNA Gel Assay

  • Agarose gel electrophoresis should be used to assess the intactness of high molecular weigh DNA.
  • Load approximately 10-20 ng of genomic DNA on to a minigel containing 1% agarose along with 50-100 ng of an appropriate molecular weight marker such as Lambda HindIII or Invitrogen 1 Kb Ladder. Run the gel for approximately 30 minutes minutes at 100V.
  • Intact genomic DNA can be viewed on this gel as a high molecular band with either minimal or absent lower molecular weight smear. The relative intensity of bands within the molecular weight ladder can be used to provide a qualitative estimate of the genomic DNA sample concentration based on relative fluorescent intensities.
  • Pricing for Sample Quality Services including Genomic DNA Gel Assay