DD/AP-PCR: combination of differential display and arbitrarily primed PCR of oligo(dT) cDNA.
In this report we describe the first direct comparison of differential display (DD) and arbitrarily primed PCR (AP-PCR) amplification of oligo(dT)-primed cDNA. Our results indicate that both of these widely used RNA fingerprinting techniques have their respective advantages and limitations. DD produces profiles specific to the anchored oligo(dT) primer used for cDNA synthesis. AP-PCR displays significant redundancy of profiles generated from different oligo(dT) cDNA pools, but is not as biased to the isolation of A/T-rich or 3' sequences. It was found that both techniques can utilize cDNA synthesized using a generic anchored oligo(dT) primer (dT12VN; equimolar amounts of dT12VA, dT12VC, dT12VG, and dT12VT, where V is dA, dC, or dG); this efficiently selects for poly(A)+ sequences from total RNA, and significantly reduces the number of cDNA preparations required per experiment. Using dT12VN cDNA pools generated from rat liver, spleen, and brain, the two approaches (AP-PCR and DD) were used in combination. Several known mRNAs were identified; some were unique to either technique and some were common to both. Since it is the RNA which is usually the limiting resource, maximum utilization may be achieved by generating a single pool of dT12VN-primed cDNA and performing both AP-PCR and DD (DD/AP-PCR).
Rothschild
C B
CB
Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157, USA.
Brewer
C S
CS
offden
D W
DW
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D051381:Rats
D017931:DNA Primers; D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; C034477:RNA primers; C027903:oligo (dT); D012313:RNA