Sample Preparation

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.

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.

DNA Isolation

The various types of DNA isolation methods are discussed in this technical note from Qiagen. 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
• meet the minimum concentration requirements
• diluted in 10mM Tris, ph 7.5 or Qiagen EB buffer

RNA Isolation

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.

When isolating RNA, a two-step purification protocol is recommended since a single organic phase extraction (i.e. Trizol) does not adequately remove proteins and DNA. The second phase of purification is usually best done through a column purification with a DNAse treatment step included. This article offers a thorough discussion of the basics of RNA isolation and tips to increase yield.

A commonly used, inexpensive RNA purification protocol is a basic Trizol extraction followed by an RNeasy column. There are also many kits available to isolate high-quality RNA. This chart offers guidelines for choosing the correct Ambion/Applied Biosystems kit for a particular sample type.

Characteristics of RNA that will perform well in GSL protocols:

• should be intact and non-degraded
• have an OD 260/280 between 1.8 - 2.0 (a value of 2.0 is generally accepted as pure RNA)
• have an OD 260/230 between 1.8 - 2.2 (this ratio is an alternative measurement of nucleic acid purity)
• have a 28S to 18S ratio of 1.0 or greater
• have a RIN (Agilent's RNA Integrity Number) of 7.0 or higher
• be pure and free of salts, proteins, DNA, or RNAses that will interfere with reactions or degrade RNA
• meet the minimum concentration requirements
• diluted in 10mM Tris, ph 7.5 or Qiagen EB buffer