Evaluation of two methods for generating cRNA for microarray experiments from nanogram amounts of total RNA.

Mads Bak; Lene Conley; Jakob Hedegaard; Lars Allan Larsen; Peter Sørensen; Christian Bendixen; Niels Tommerup

doi:10.1016/j.ab.2006.08.023

Evaluation of two methods for generating cRNA for microarray experiments from nanogram amounts of total RNA.

Mads Bak, Lene Conley, Jakob Hedegaard, Lars Allan Larsen, Peter Sørensen, Christian Bendixen, Niels Tommerup

5 Citations (Scopus)

Abstract

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.
Udgivelsesdato: 2006-Nov-1

Original language	English
Journal	Analytical Biochemistry
Volume	358
Issue number	1
Pages (from-to)	111-9
Number of pages	8
ISSN	0003-2697
DOIs	https://doi.org/10.1016/j.ab.2006.08.023
Publication status	Published - 2006

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10.1016/j.ab.2006.08.023

Cite this

@article{09fc19a00c4b11ddbee902004c4f4f50,

title = "Evaluation of two methods for generating cRNA for microarray experiments from nanogram amounts of total RNA.",

abstract = "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. Udgivelsesdato: 2006-Nov-1",

author = "Mads Bak and Lene Conley and Jakob Hedegaard and Larsen, {Lars Allan} and Peter S{\o}rensen and Christian Bendixen and Niels Tommerup",

note = "Keywords: Animals; Liver; Lung; Nucleic Acid Amplification Techniques; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; RNA; RNA, Complementary; Reproducibility of Results; Swine; Transcription, Genetic",

year = "2006",

doi = "10.1016/j.ab.2006.08.023",

language = "English",

volume = "358",

pages = "111--9",

journal = "Analytical Biochemistry",

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publisher = "Elsevier",

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TY - JOUR

T1 - Evaluation of two methods for generating cRNA for microarray experiments from nanogram amounts of total RNA.

AU - Bak, Mads

AU - Conley, Lene

AU - Hedegaard, Jakob

AU - Larsen, Lars Allan

AU - Sørensen, Peter

AU - Bendixen, Christian

AU - Tommerup, Niels

N1 - Keywords: Animals; Liver; Lung; Nucleic Acid Amplification Techniques; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; RNA; RNA, Complementary; Reproducibility of Results; Swine; Transcription, Genetic

PY - 2006

Y1 - 2006

N2 - 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. Udgivelsesdato: 2006-Nov-1

AB - 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. Udgivelsesdato: 2006-Nov-1

U2 - 10.1016/j.ab.2006.08.023

DO - 10.1016/j.ab.2006.08.023

M3 - Journal article

C2 - 16996470

SN - 0003-2697

VL - 358

SP - 111

EP - 119

JO - Analytical Biochemistry

JF - Analytical Biochemistry

IS - 1

ER -