Scripps Research Logo

Microarray Core Facility

DNA/RNA Isolation

The Microarray Core offers RNA extraction as a service. Preparation of DNA/RNA for microarray analysis is a critical step and one of the most significant sources of variability in microarray data. DNA extraction should yield high molecular weight minimally degraded sample for best results. Successful RNA extraction will yield total RNA with minimal degradation and free of any contaminating RNAses.

Please consult with Microarray Core personnel regarding the scope of your planned experiment in order to best choose the extraction method best suited for your samples. Addiotnally, we advise consulting technical support and protocols from reagent companies well versed in nucleic acid purification (Life Technologies, Qiagen, Zymo). 

RNA Preservation:

It is critical that messenger RNA be preserved in its last functioning, physiological state and be prevented from degrading. This is achieved by one of two methods for tissues: snap freezing in liquid nitrogen or emersion in RNA Later. The liquid nitrogen process is particularly suitable for larger tissue samples ( >50mg). Tissues of this size are easily visualized and ground by a liquid nitrogen cooled mortar and pestle (Fisher) with negligible loss during processing. Smaller tissue samples, such as needle core biopsies or murine lymph nodes, are more suitable to preservation in RNA Later solution, a product distributed by both Ambion and Qiagen. This product is designed to penetrate cell membranes and inactivate cellular and other contaminating RNAses. Tissue pieces must be no greater than 5mm in thickness in order to allow adequate infiltration by the RNA Later solution and effectively prevent degradation. Small tissue samples preserved in RNA Later can be homogenized in tissue grinders (Fisher).

When working with cell preparations such as purified lymphocytes, pelleting the cells and resuspending the pellet in a chaotropic agent most effectively accomplishes both the preservation and recovery of cellular RNA. (Trizol or RLT buffers are commonly used for this purpose.) After suspension in the chaotropic agent, the cellular material can be homogenized using a QiaShredder column and the RNA extracted using an RNeasy column.

Regardless of the method used to extract the total cellular RNA, we have observed a significant improvement in yields of cDNA synthesized from RNA extracted using protocols that include a final column purification step (e.g. RNeasy). We believe this final "washing" step may remove residual components from the RNA that may inhibit one or more of the enzymes used in cDNA synthesis. In addition, a DNAse digestion step may be performed on the extracted RNA if gel analysis reveals significant contaminating genomic DNA. Contaminating DNA can interfere with accurate quantitation of RNA.

Shipping DNA/RNA Samples

DNA samples can be shipped in TE Buffer on wet ice or lyophilized with no refridgeration. RNA can be shipped on dry ice. Both DNA and RNA can be shipped at ambient temperatures following stabilization in RNAstable/DNAstable (Biometrica).

DNA/RNA Quantitation and Quality:

The Microarray Core uses Nanodrop, Qubit, and BioAnalyzer technologies in order to asses the quantity and quality of purified DNA/RNA upon receipt of samples. All samples are evaluated prior to beginning any genomic experiments.