A sensitive transcriptome analysis method that can detect unknown transcripts.
We have developed an AFLP-based gene expression profiling method called 'high coverage expression profiling' (HiCEP) analysis. By making improvements to the selective PCR technique we have reduced the rate of false positive peaks to approximately 4% and consequently the number of peaks, including overlapping peaks, has been markedly decreased. As a result we can determine the relationship between peaks and original transcripts unequivocally. This will make it practical to prepare a database of all peaks, allowing gene assignment without having to isolate individual peaks. This precise selection also enables us to easily clone peaks of interest and predict the corresponding gene for each peak in some species. The procedure is highly reproducible and sensitive enough to detect even a 1.2-fold difference in gene expression. Most importantly, the low false positive rate enables us to analyze gene expression with wide coverage by means of four instead of six nucleotide recognition site restriction enzymes for fingerprinting mRNAs. Therefore, the method detects 70-80% of all transcripts, including non-coding transcripts, unknown and known genes. Moreover, the method requires no sequence information and so is applicable even to eukaryotes for which there is no genome information available.
Fukumura
Ryutaro
R
Transcriptome Profiling Group, National Institute of Radiological Sciences, Anagawa 4-9-1, Inage-ku, Chiba-shi, Chiba 263-8555, Japan.
Takahashi
Hirokazu
H
Saito
Toshiyuki
T
Tsutsumi
Yoko
Y
Fujimori
Akira
A
Sato
Shinji
S
Tatsumi
Kouichi
K
Araki
Ryoko
R
Abe
Masumi
M
D000818:Animals; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger