Direct quantification of HIV-1 RNA by capillary electrophoresis with laser-induced fluorescence.
HIV-1 RNA was quantitated directly by capillary electrophoresis with laser-induced fluorescence (CE-LIF). CE-LIF was used to analyze cellular RNA and various nucleotide complexes. A fluorescently labeled DNA probe (DNA/RNA complex) in conjunction with thiazole orange intercalator was determined to have optimal stability and sensitivity for RNA analysis. Based on this observation, a hybridization method using a HIV-specific fluorescently labeled probe with analysis by CE-LIF was developed. Plasma samples from a HIV-seropositive patient were lysed to obtain RNA, hybridized with the HIV-specific probe and analyzed by CE-LIF. As little as 19 fg (1710 copies per 1 ml of starting plasma) of HIV RNA can be reliably and quantitatively detected. CE-LIF appears to be an efficient and sensitive method to quantitatively analyze RNA from a variety of sources.
Kolesar
J M
JM
School of Pharmacy, University of Wisconsin, Madison 53706-1515, USA.
Allen
P G
PG
Doran
C M
CM
D000818:Animals; D052160:Benzothiazoles; D015342:DNA Probes; D019075:Electrophoresis, Capillary; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005814:Genes, Viral; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D007364:Intercalating Agents; D007834:Lasers; D009711:Nucleotides / Q000032:analysis; D011804:Quinolines; D012313:RNA / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D012367:RNA, Viral / Q000097:blood* / Q000235:genetics; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D018411:Spodoptera / Q000235:genetics; D013844:Thiazoles
D052160:Benzothiazoles; D015342:DNA Probes; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D009711:Nucleotides; D011804:Quinolines; D012367:RNA, Viral; D013844:Thiazoles; C051000:thiazole orange; D012313:RNA