MEASURING mRNA QUALITY

Christine L. Miller¹, Suad Diglisic², Flora Leister¹,

Maree Webster²,

1Stanley

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,

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

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