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Normalization of Circulating Nucleic Acid Results
Author(s) -
Holford N.C.,
Ramoutar A.,
Butt A.N.,
Swaminathan R.
Publication year - 2008
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1196/annals.1448.000
Subject(s) - gene expression , thyroid , messenger rna , complementary dna , medicine , endocrinology , real time polymerase chain reaction , actin , hormone , biology , gene , microbiology and biotechnology , biochemistry
Nucleic acids, both DNA and mRNA, have been detected in the circulation and have been demonstrated to be useful in such areas as fetal medicine, oncology, and transplantation. When mRNA is measured in circulating blood, the results are expressed in relation to a reference gene product in order to correct for any differences in extraction, volume of starting material or other differences. Many authors use β‐actin mRNA and express results as a ratio of target mRNA to β‐actin mRNA. We have used a similar approach when studying diabetic retinopathy. Recently, we planned to investigate the expression of thyroid dependent gene expression in acutely ill patients. As a control study, we examined the expression of thyroid hormone–dependent gene expression in subjects with hyperthyroidism and found that the expression of β‐actin mRNA was affected by thyroid hormone status. Blood samples were taken into PAX gene™ tubes from 31 healthy subjects (mean age, 43 ± 16 yrs) and 7 patients with hyperthyroidism (mean age, 43 ± 5 yrs). Diagnosis of hyperthyroidism was confirmed by clinical findings and biochemical results. After extraction of mRNA, cDNA was synthesized using reverse transcription. Quantification of Na/K‐ATPase, T3 receptor, and β‐actin cDNA was carried out by RT‐PCR. Median β‐actin levels were significantly higher in hyperthyroid subjects compared to healthy subjects (18.2 versus 2.30; P < 0.00042). When mRNA for the T3 receptor was expressed in relation to β‐actin, there was a significantly higher in hyperthyroidism (0.0168 versus 0.218, P < 0.05). However, this was significantly lower when expressed in relation to total RNA (12.2 versus 2.24, P < 0.00015).We conclude that normalizing results to β‐actin may not be appropriate in all circumstances.