Combining the technique of RNA fingerprinting and differential display to obtain differentially expressed mRNA.
We have modified recently developed methods of RNA fingerprinting and differential display based on arbitrarily primed PCR which can be used for detection and cloning of differentially expressed mRNAs. Our protocol requires only a single cDNA synthesis for each different RNA sample, in contrast to the multiple cDNA reactions required for differential display method, followed by selective amplification of cDNA sequence fraction by arbitrary and oligo(dT) primers. We have shown that the longer primers (25-29-mers) allow the use of optimal dNTP concentration and higher stringency PCR. Further improvements include using TaqStart antibody for hot start PCR and thermostable enzyme mixes suitable for long-distance PCR. Long-distance PCR enables the method to display bands of up to 2 kb and should result in a higher fidelity of PCR products to the original RNA template. When these improvements are combined the resulting method is highly reproducible, and more than 85% of the differentially expressed bands can be confirmed by Northern blot analysis.
Diachenko
L B
LB
CLONTECH Laboratories, Inc., Palo Alto, California 94303-4230, USA.
Ledesma
J
J
Chenchik
A A
AA
Siebert
P D
PD
D000328:Adult; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D017931:DNA Primers; D018076:DNA, Complementary; D005333:Fetus; D005821:Genetic Techniques; D006801:Humans; D008099:Liver / Q000196:embryology / Q000378:metabolism; D008322:Mammals; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000737:chemistry*; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000737:chemistry*; D013698:Templates, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D012313:RNA