Christine L. Miller1, Suad Diglisic2, Flora Leister1,

Maree Webster2,

Robert H. Yolken1


Division of Developmental Neurovirology, Department of Pediatrics, Johns Hopkins

University, Baltimore, MD 21287 and 2The Stanley Medical Research

Institute, Uniformed Services University of the Health Sciences, Bethesda, MD,

20814, USA



The ability to quickly

assess mRNA quality using small amounts of total RNA has become increasingly

important as the subsequent measures of mRNA transcripts have become more

comprehensive and more expensive.  Some screening ability is particularly

crucial when working with postmortem human tissue, which exhibits variable

degrees of mRNA degradation.  In the past, evaluating mRNA quality in total

human RNA preparations relied heavily on estimating the ratio of the 28S to 18S

ribosomal components seen when the RNA was subjected to gel electrophoresis. 

This approach evolved because the ribosomal bands were the most clearly visible

components of the total RNA.  Within the past few years, it has become possible

to utilize microfluidic electrophoresis coupled with sensitive fluorescence

detection to generate a complete elution profile of the total RNA sample. This

feature has allowed us to re-examine the relationship between the total RNA

profile and mRNA quality.


We assessed mRNA quality in

105 total RNA extracts of postmortem human brain tissue by utilizing random

hexamer primed-reverse transcription followed by real-time PCR for 4

housekeeping genes.  The microfluidic elution/electropherogram profile of the

total RNA was carried out on an Agilent Bioanalyzer.  Comparison of the mean

housekeeping gene score with seven different sections of the RNA

electropherogram revealed three main findings: (1) There was no significant

correlation between the ration of the ribosomal 28S and 18S peak areas to the

mRNA housekeeping gene score (r= -0.06, n .s.). (2) The most significant

correlation between the two methods was between the sum of [percent of total

area occupied by the 18S peak with underlying baseline] plus [percent of total

area occupied by the RNA eluting between the 18S and 28S peaks], r=0.41,

p=0.0001.  The area of the electropherogram encompassed by this measure overlaps

to a large degree with the predicted elution time of intact mRNA.  Neither

measure, (1) or (2) of the electropherogram, allowed a satisfactory

discrimination of the RNA by housekeeping gene score, since a threshold could

not be set which would adequately minimize false positives and false negatives. 

Thus, (3) a measure was identified which did allow the discrimination of the

lowest housekeeping score samples, specifically the ratio of the 18S peak height

to the highest peak in the t-RNA to 18S rRNA interval.  If the threshold for

that value was set at 2.12, the six lowest housekeeping score samples could be

discriminated from the remainder of the sample set.