Evaluation of two methods for generating cRNA for microarray experiments from nanogram amounts of total RNA.
Several methods have been developed for amplification of RNA, making it possible to use cDNA microarrays for analysis of samples limited in amount of total RNA. The most widely used amplification protocol, the Eberwine method, amplifies RNA in a linear manner through in vitro transcription (IVT). However, when starting material is limited to nanogram amounts of total RNA, several rounds of amplification are necessary, making this method both expensive and labor-intensive. Amplification by PCR is robust and is able to amplify extremely limiting material. However, it is possible that the nonlinear nature of PCR could result in reduced reproducibility of the amplification compared with IVT. We have evaluated two methods that use a combination of PCR and IVT for amplification of nanogram amounts of total RNA. We have compared microarray results obtained by these methods with results obtained by two established methods: indirect labeling of 20 microg total RNA and Eberwine amplification of 1 microg total RNA. Starting from as little as 5 ng of total RNA, both methods yielded results in concordance with the Eberwine method.
Bak
Mads
M
Wilhelm Johannsen Center for Functional Genome Research, Institute of Medical Biochemistry and Genetics, Panum Institute, University of Copenhagen, DK-2200N Copenhagen, Denmark. mads@imbg.ku.dk
Conley
Lene
L
Hedegaard
Jakob
J
Larsen
Lars Allan
LA
Sørensen
Peter
P
Bendixen
Christian
C
Tommerup
Niels
N
D000818:Animals; D008099:Liver / Q000737:chemistry; D008168:Lung / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000302:isolation & purification; D018075:RNA, Complementary / Q000302:isolation & purification*; D015203:Reproducibility of Results; D013552:Swine; D014158:Transcription, Genetic
D018075:RNA, Complementary; D012313:RNA