Optimization of fragmentation conditions for microarray analysis of viral RNA.
An important consideration in microarray analysis of nucleic acids is the efficiency with which the target molecule is captured by, or hybridized to, surface-immobilized oligos. For RNA, secondary and tertiary structure of the target strand can significantly decrease capture efficiency. To overcome this limitation, RNA is often fragmented to reduce structural effects. In this study, the metal ion-catalyzed base hydrolysis fragmentation conditions for viral RNA extracted from influenza viruses were evaluated and the hybridization efficiency of the resulting fragments was determined as a function of fragment length. The amount of RNA captured was evaluated qualitatively by fluorescence intensity normalized to an internal standard. Optimized conditions for influenza RNA were determined to include a fragmentation time of 20-30 min at 75 degrees C. These conditions resulted in a maximum concentration of fragments between 38 and 150 nt in length and a maximum in the capture and label efficiency.
Mehlmann
Martin
M
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA.
Townsend
Michael B
MB
Stears
Robin L
RL
Kuchta
Robert D
RD
Rowlen
Kathy L
KL
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D006358:Hot Temperature; D009980:Influenza A virus / Q000235:genetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics* / Q000302:isolation & purification
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral