A quantitative polymerase chain reaction assay for interleukin 5 messenger RNA.
The quantitation of low abundance messenger (m)RNA species such as that for interleukin 5 (IL-5) cannot be achieved by Northern blotting. Polymerase chain reaction (PCR) technology can be used to overcome this paucity of target molecules, but in its standard format it is semiquantitative. We describe the synthesis, purification, and use of a synthetic IL-5 mRNA (cRNA) molecule which, when included in the PCR reaction, enabled the amplification of IL-5 mRNA molecules from clinical samples to be made quantitative. A stringent protocol involving analysis of the purified transcript by PCR (to ensure its freedom from template molecules) is required. Preliminary experiments indicate that IL-5 mRNA levels are elevated in asthmatics.
Guiffre
A
A
Department of Hematology, St. Vincent's Hospital, Rushcutter's Bay, Australia.
Atkinson
K
K
Kearney
P
P
D001483:Base Sequence; D015152:Blotting, Northern; D004247:DNA / Q000235:genetics; D005069:Evaluation Studies as Topic; D006801:Humans; D015848:Interleukin-5 / Q000235:genetics*; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D018075:RNA, Complementary; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D012680:Sensitivity and Specificity
D015848:Interleukin-5; D018075:RNA, Complementary; D012333:RNA, Messenger; D012313:RNA; D004247:DNA