Optimized RNA amplification using T7-RNA-polymerase based in vitro transcription.
The use of expression profiling to explore a cell's transcriptional landscape has exploded in recent years. In many cases, however, the very limited amount of starting material poses a major problem, making the amplification of the isolated RNA obligatory. The most prominent amplification method used was developed by the Eberwine lab in 1990: cDNA synthesis is started with an oligo(dT) primer containing a T7 RNA polymerase promoter. After second-strand synthesis RNA is transcribed in vitro using T7 RNA polymerase. It has been demonstrated that antisense RNA amplification not only preserves the fidelity of RNA-based microarray analysis but even improves the sensitivity. In our aim to improve the yield of in vitro transcription reactions and to facilitate the use of amplified RNA for the construction of cDNA libraries we tested a series of T7 primers with different 3' flanking sequences containing restriction sites. In addition we tested the impact of different DNA polymerases used for synthesizing the templates on the efficiency of the in vitro transcription reaction. A total of 28 different oligo(dT)-T7 promoter primers were tested. Two of them showed a dramatically increased yield of RNA from the in vitro transcription reaction. The combination of the improved second-strand synthesis with the new T7 primer increased the RNA yield 60-fold compared to the yield of standard procedures.
Moll
Pamela R
PR
Fachbereich Zellbiologie, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria.
Duschl
Jutta
J
Richter
Klaus
K
D024509:3' Flanking Region; D002474:Cell-Free System; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D015723:Gene Library; D021141:Nucleic Acid Amplification Techniques; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins
D017931:DNA Primers; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases