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`PAGE 5 OF 9
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`  
`
`40-
`
`35-
`
`30
`
`M {J1
`
`Intensitymm M G
`
`15-
`
`10
`
`Degraded RNA
`
`
`

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`
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`
`Nucleic Acids Research, 2000, Vol. 29, No. 2 e7
`
`1000 ng
`
`/ 500 I10
`
`
`
`
`
`
`Retention Time (min)
`
`11100000 *
`
`1200000
`
`1000000
`
`800000
`
`600000 -'
`
`400000 --
`
`200000 -
`
`
`IntegratedPeakarea[mVsl
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` !"#$%&'()*+,-./ 0)1&0&23,+%+%&%42567%61,*89+):9-2,>,2=)07&)?;3&5&*)7+=5&214<)-=)0*600&*+,6-#$%&75&;&-*&)-2,-+&:5,+46@*)7+=5&2'()*+,-./ 16+%=-0)(
`;%63;):5&)+&55&+&-+,6-+,<&+%)-=-0)1&0&2'()*+,-./ #
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`
`0
`
`200
`
`400
`
`500
`
`800
`
`1000
`
`1200
`
`M52 RNA concentration [ngj
`
`Figure 4. RNA quantification after IP RP HPLC by peak integration. (A) Superimposed chromatograms of known amounts of M52 RNA analyzed by 1? RP HPLC
`under gradient condition 2. (B) Standard curve for MSZ RNA quantification, plot of integrated peak area from (A) versus injected amount of M52 RNA (Table 1). The
`square of the Pearson product moment correlation coefficient (R2) through the given data points is 0.9985.
`
`(9,10). The B-actin RNA labeled with the hydrophobic DIG tag
`shows a greater retention time than unlabeled B—actin RNA.
`Individual peaks were captured by manual collection. The
`
`presence and integrity of captured B-actin RNA, both unla-
`beled and labeled, was confirmed by reverse transcription—PCR
`using 5 pl aliquots of each collected sample. A 550 bp PCR
`
`5
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`
`7
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`PAGE 8 OF 9
`
`
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`e7 Nucleic Acids Research, 2000, Vol. 29, No. 2
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`
`
`
`
`Intensity[mV}
`
`0
`
`S
`
`10
`
`15
`
`20
`
`25
`
`Retention Time (min)
`
`50 .
`
`
`
`45
`
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`
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`
`D
`
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`
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`
`Figure 6. (Previous page and above) Mouse brain total RNA analyzed by IP RP HPLC. All RNA samples were analyzed by IP RP HPLC using gradient condition 1.
`(A) Mouse brain total RNA (20 pg). (B) Analysis of large rRNAs (a mixture of co—eluting 285 and 185 rRNA, respectively) purified by peak capture from total RNA.
`(C) Analysis of mRNA purified by peak capture from total RNA. (D) Analysis of mouse brain poly(A)+ mRNA (5 pg) obtained after two rounds of oligo(d'l‘)—cellulose
`purification.
`
`used here the two rRNA peaks are not well resolved, i.e. the
`peaks for the 288 and 188 rRNA show significant overlap.
`Thus, the rRNA peaks in Figure 6A and B are labeled as large
`rRNAs.
`
`For comparative purposes mouse brain mRNA (Clontech),
`which was isolated by two rounds of poly(A)+ RNA selection,
`was analyzed by IP RP HPLC. As shown in Figure 6D, this
`
`sample still contained some rRNA contamination (confirmed
`by reverse transcription—PCR analysis) after two rounds of
`poly(A)+ selection. Significantly less rRNA contamination is
`observed after only a single round of purification by IP RP
`HPLC (Fig. 7C). Recovery of RNA after IP RP HPLC is generally
`higher than that after oligo(dT)—cellulose purification. Purification
`of RNA samples by IP RP HPLC can be performed in a matter
`
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