Sample Preparation


Sample Requirements | Isolation | Quality Control | Submitting Samples

Sample quality is the single most important factor in the success of a genomics experiment. The higher the quality of input sample; the higher the quality of output data. Any isolation protocol or product may be used that results in pure, intact nucleic acid free of excess salts or proteins. A methodology routinely used in a customer’s lab that has produced high quality DNA in the past will be more reliable than any newly purchased kit or downloaded protocol.

GSL Isolation of Nucleic Acids


The GSL accepts a variety of material for DNA or RNA isolation, including blood, saliva, cells, tissue, buccal swabs, and fecal matter. Tubes containing material for isolation must be clearly labeled with the GSL project and sample number ("1234-SL-0001"). Please disucss your project goals with us before submission of material as we can advise on the best isolation procedures to be used for the experiment.

DNA Isolation

There are many methods for isolation of DNA that will yield high quality DNA
  • Isolation methods are dependent on sample type, sample input and final application
  • For commercially available kits please see Ambion or Qiagen for product lists
  • Other methods may be acceptable if the final samples meet our basic guidelines (see below)
  • The following DNA extraction/purification methods have yielded good results for GSL customers:
    • SDS/ProK digestion, phenol-chloroform extraction, Microcon® or Centricon® (Millipore) ultrapurification and concentration
    • QIAGEN; QIAmp® DNA Blood Maxi Kit
    • (Targeted Genotyping Only) Gentra PUREGENE
    • PAXgene Blood DNA Kits

Characteristics of DNA that will perform well in GSL protocols:
  • Should be double-stranded and intact (non-degraded)
  • Have an OD 260/280 between 1.8 - 2.0
  • Be pure and free of salts, proteins, and/or DNAses that will interfere with reactions or degrade DNA
  • Be free of RNA (RNAse treatment should be performed as part of the DNA isolation
  • Meet the minimum concentration requirements
  • Diluted in 10mM Tris, ph 7.5 or Qiagen EB buffer
  • If submitted DNA is too dilute to use in the protocol, we will perform a bead purification protocol to concentrate the DNA for an additional fee.

GSL services include QC of all submitted samples prior to processing. If samples fail our QC metrics you will be contacted. You may choose to send replacement samples or proceed with the failed samples. Please note that we cannot guarantee successful library preparation or sequencing results for samples that have failed our QC metrics.

Visualization of gDNA by gel. The gel image on the left shows high-quality, high-molecular weight, intact gDNA in all wells. The gel image on right shows several samples that would not pass GSL QC. Sample A1 shows a smear of gDNA indicating degradation. Sample A2 also shows intact gDNA, but as evidenced by the excess sample remaining in the well, the sample was provided at a concentration much higher than requested. Sample B2 shows no intact gDNA. Samples B1 and A3 show high-quality gDNA that would perform well in GSL protocols.

Agilent 2100 Bioanalyzer electropherograms for two RNA samples.

The electropherogram on top shows good-quality RNA with a 28S/18S rRNA ratio of 1.7 and an RNA Integrity Number of 8.9 (max is 10.0). Note the rRNA peaks are distinct, with 28S higher than the 18S, and there are only a few smaller peaks that indicate RNA degradation. This sample should perform well in GSL protocols.

The electropherogram on the bottom shows poor-quality RNA with a RIN of 6. The 28S rRNA peak is shorter than the 18S peak and many smaller peaks are seen along the baseline, indicating a fair amount of RNA degradation. This sample would not pass GSL QC requirements for use in most protocols.

RNA Isolation and Quality Control

Please note that all of our RNA services require submission of total RNA.

There are many methods for isolation of total RNA that will yield high quality RNA.
  • Isolation methods are dependent on sample type, sample input and final application
  • For commercially available kits please see Ambion or Qiagen for product lists
  • Other methods may be acceptable if the final samples meet our basic guidelines (see below)
*Please note that total RNA that is submitted for small RNA-Seq needs to be isolated using a method that retains small RNA.

Basic guidelines for high quality total RNA samples that will perform well in GSL protocols:
  • Intact
  • 28S to 18S ratio of 1.0 or greater
  • RIN (Agilent's RNA Integrity Number) of 7.0 or higher
  • Free of salts, proteins, DNA, and RNAses
    • OD 260/280 between 1.8 - 2.0 (a value of 2.0 is generally accepted as pure RNA)
    • OD 260/230 between 1.8 - 2.2 (this ratio is an alternative measurement of nucleic acid purity, lower values indicate contamination)
  • Resuspended in a suitable buffer
  • Meet GSL minimum concentration requirements

GSL services include QC of all submitted samples prior to processing. Some quality measures such as RIN and 28S to 18S ratio are based on certain species (human, mouse, rat) and other organisms’ values for those metrics may differ. For example, insect bioanalysis profiles may not generate good RIN numbers, but still be high quality, intact RNA that will perform well in protocols. You should find the expected profile for your species and assess the quality by comparing our bioanalysis results to that profile. When an RNA profile is atypical, we require your approval before continuing with the sample. If samples fail our QC metrics you will be contacted. You may choose to send replacement samples or proceed with the failed samples. Please note that we cannot guarantee successful library preparation or sequencing results for samples that have failed our QC metrics.