aRNA-longSAGE: a new approach to generate SAGE libraries from microdissected cells.
Large-scale gene expression analyses of microdissected primary tissue are still difficult because generally only a limited amount of mRNA can be obtained from microdissected cells. The introduction of the T7-based RNA amplification technique was an important step to reduce the amount of RNA needed for such analyses. This amplification technique produces amplified antisense RNA (aRNA), which so far has precluded its direct use for serial analysis of gene expression (SAGE) library production. We describe a method, termed 'aRNA-longSAGE', which is the first to allow the direct use of aRNA for standard longSAGE library production. The aRNA-longSAGE protocol was validated by comparing two aRNA-longSAGE libraries with two Micro-longSAGE libraries that were generated from the same RNA preparations of two different cell lines. Using a conservative validation approach, we were able to verify 68% of the differentially expressed genes identified by aRNA-longSAGE. Furthermore, the identification rate of differentially expressed genes was roughly twice as high in our aRNA-longSAGE libraries as in the standard Micro-longSAGE libraries. Using our validated aRNA-longSAGE protocol, we were able to successfully generate longSAGE libraries from as little as 40 ng of total RNA isolated from 2000-3000 microdissected pancreatic ductal epithelial cells or cells from pancreatic intraepithelial neoplasias.
Heidenblut
Anna M
AM
Department of Internal Medicine, Knappschaftskrankenhaus, University of Bochum, Bochum, Germany.
Lüttges
Jutta
J
Buchholz
Malte
M
Heinitz
Christian
C
Emmersen
Jeppe
J
Nielsen
Kåre Lehmann
KL
Schreiter
Pat
P
Souquet
Manfred
M
Nowacki
Sandra
S
Herbrand
Ulrike
U
Klöppel
Günter
G
Schmiegel
Wolff
W
Gress
Thomas
T
Hahn
Stephan A
SA
D018938:Caco-2 Cells; D018076:DNA, Complementary / Q000096:biosynthesis; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D042282:Microdissection; D021141:Nucleic Acid Amplification Techniques; D010179:Pancreas / Q000166:cytology / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D016133:Polymerase Chain Reaction; D012313:RNA / Q000737:chemistry; D016372:RNA, Antisense / Q000096:biosynthesis*
D018076:DNA, Complementary; D016372:RNA, Antisense; D012313:RNA