CHARACTERISTICS OF RNA THAT AFFECT cDNA GENERATION AND ARRAYHYBRIDIZATION

array hybridizations that utilize different methodologies.  At least some

of the disparity may be attributable to the relative ability to access distinct

pools of RNA.  We have compared two different methods of RNA extraction,

and two methods of oligonucleotide priming, in order to optimize the yield and

range of cDNA templates that can be generated from post-mortem brain

tissue.  The cDNA product is influenced by the overall quality of the RNA

starting material, as well as structural characteristics of specific RNA species

that influence the ability of reverse transcriptase to carry out its

function.  From the same pool of homogenized brain tissue, the Trizol

method of extraction resulted in a lower quality of RNA than did the Qiagen

method, as visualized on an agarose gel and reflected in quantification of

RT-PCR products.  In addition, the RNA  extracted by Trizol was

markedly less stable at 37°C.  Although both methods of extraction yielded

RNA that primed better with random hexamers (RH) than with oligo-dT (dT), the

difference between priming with dT and priming with RH was much greater for the

Trizol product than the Qiagen product.  Data will be presented to show

that such differences in priming likely result from structural features specific

to certain RNAs, structures which appear to be disrupted by the Qiagen column

the Qiagen extract that do not prime with dT but prime very well with RH. 

This finding would predict that array methodologies that rely on RH priming

would detect a greater percentage of targets than those that rely on dT

priming.  Other points to be considered include the practice of pooling

samples, either at the RNA or cDNA stage, which could theoretically lead to

strong interactions between sense template present in one sample and antisense

template present in another sample. The methods of RNA extraction and probe

generation may be important variables in the performance and interpretation of