How to judge the quality of extracted chromosomal RNA samples

Complete total RNA will produce clear 28S and 18S rRNA bands (eukaryotic samples) when subjected to denaturing gel electrophoresis. The intensity of the 28S rRNA band should be approximately twice that of the 18S rRNA band. This 2:1 ratio (28S:18S) is a good indicator of RNA integrity.

One drawback to using denaturing gel electrophoresis to assess RNA integrity is the amount of RNA sample required for observation. Typically, at least 200 ng of RNA needs to be loaded in a denaturing gel to be visualized under EtBr staining. In some RNA preparation experiments, such as extracting RNA from needle biopsy samples or laser capture microdissection samples, the yield is very low. In this case, it may not be possible to remove 200ng of RNA to assess its integrity before performing expression profiling experiments. Other nucleic acid dyes, such as Moleculor Probes (Eugene, OR)'s SYBR? Gold and SYBR Green II RNA dyes, can significantly improve sensitivity compared to traditional EtBr staining methods for agarose gel electrophoresis. By using a 300nm transmitted light source (6 x 15-watt bulbs) and special filters, the SYBR Gold RNA gel stain can detect as little as 1ng of RNA, while SYBR Green II can detect as little as 2ng, allowing the use of larger gels. Small samples are required for RNA integrity analysis.

There is currently a fast and simple method that can replace traditional gel analysis methods, which can simultaneously quantify and evaluate RNA samples. The Agilent 2100 Bioanalyzer (Agilent Technologies) is the first commercially available microfluidic instrument to provide detailed information on the status of RNA samples. When used in conjunction with the RNA 6000 LabChip? (a registered trademark owned by Caliper Technologies Corporation), as little as 1μl of sample at a concentration of 10 ng/μl is required for each analysis. In addition to assessing RNA integrity, this automated system can also provide assessment of sample RNA concentration and purity (such as rRNA contamination in mRNA preparation). In the past, testing concentration and purity required using a portion of the RNA sample (via A260 spectrophotometry), and assessing integrity required using an additional portion of the sample. By using the LabChip? system, concentration, integrity and purity can be analyzed simultaneously using only a 5ng sample. Analysis data can be displayed in gel-like images, electrophoresis peak diagrams, tables, etc.