A method for gene expression analysis by oligonucleotide arrays from minute biological materials

Ji, Wan; Zhou, Wenli; Gregg, Keqin; Lindpaintner, Klaus; Davis, Sara K.; Davis, Scott

doi:10.1016/j.ab.2004.03.039

Cited by 32 publications

(28 citation statements)

References 25 publications

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“…We took advantage of a SMART PCRcDNA approach that combines PCR amplification and T7 RNA polymerase to amplify submicrogram RNA samples (Ji et al, 2004). Although some distortion of within-sample stoichiometry occurs with this method, one can assume the same distortion between samples, thus maintenance of between-sample stoichiometry allows comparative analyses.…”

Section: Transcriptional Profiling Of Nccit Cells Relative To 293t Cellsmentioning

confidence: 99%

Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells

Taranger

Noer

Sørensen

et al. 2005

MBoC

254

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Section: Transcriptional Profiling Of Nccit Cells Relative To 293t Cellsmentioning

confidence: 99%

Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells

Taranger

Noer

Sørensen

et al. 2005

MBoC

254

199

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“…RSKs are activated through direct phosphorylation by the mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) in response to insulin and growth factors, oncogenic events, and ultraviolet irradiation. RSKs have been implicated in the stimulation of cell proliferation and differentiation, in the cellular stress response, and in apoptosis (5 ). To date, only a few RSK2-specific physiologic substrates have been described: the transcription factor CREB, histone H3, STAT3 (6 ), ATF4 (7 ), and p53 (8 ).…”

Section: Identification Of Novel Mutations In Patients Withmentioning

confidence: 99%

“…Coffin-Lowry Syndrome by a Denaturing HPLC-Based Assay, Michele Falco, 1 Corrado Romano, 2 Antonino Alberti, 2 Donatella Greco, 2 Carmela Scuderi, 3 Emanuela Avola, 2 Pinella Failla, 2 Serena Belli, 4 John L. Tolmie, 5 Silvestra Amata, 1 Coffin-Lowry syndrome (CLS; MIM #303600) is characterized by learning difficulties and dysmorphic traits in male patients and in some female carriers of the Xchromosome-linked gene mutation. The dysmorphic traits, skeletal abnormalities, and other clinical findings have been described (1 ).…”

Section: Identification Of Novel Mutations In Patients Withmentioning

confidence: 99%

“…The cRNA was then hybridized to the human GeneChip H133A (Affymetrix) as described previously (5 ). The numbers of genes detected as present (ϳ30%) were comparable among arrays prepared with various starting amounts of total RNA.…”

mentioning

confidence: 99%

“…The SMART (switching mechanism at the 5Ј end of RNA templates of reverse transcriptase) method (1,2 ), in combination with PCR, has been used to amplify minute samples obtained from sources such as laser capture microdissections and biopsies for gene expression analysis using oligonucleotide microarray gene chip technology (3)(4)(5)(6)(7). Amplifications Ͼ10 5 -fold can be achieved in a short time.…”

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Amplification of Nanogram Amounts of Total RNA by the SMART-Based PCR Method for High-Density Oligonucleotide Microarrays

Zhou¹,

Abruzzese²,

Polejaeva³

et al. 2005

Clinical Chemistry

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The SMART (switching mechanism at the 5Ј end of RNA templates of reverse transcriptase) method (1, 2 ), in combination with PCR, has been used to amplify minute samples obtained from sources such as laser capture microdissections and biopsies for gene expression analysis using oligonucleotide microarray gene chip technology (3-7 ). Amplifications Ͼ10 5 -fold can be achieved in a short time. However, reports in the literature have rarely discussed the feasibility and reproducibility of PCR-based methods when the amount of starting total RNA in a sample is only a very few nanograms or less, which is often the case for clinical samples.We developed a modified SMART-based PCR protocol with which reliable and robust gene expression data can be obtained from 1 ng of starting total RNA. We also investigated sources of array data variations.The modified protocol (see Supplement 1 in the Data Supplement that accompanies the online version of this Technical Brief at http:www.clinchem.org/content/vol51/ issue12) has three major changes compared with the published standard procedure (5 ): the volume of the reverse transcription reaction is reduced to 5 L; 50 ng of poly(dG/dC) carrier (Sigma) is added to the reverse transcription reaction; and PowerScript reverse transcriptase (BD Biosciences Clontech) is used instead of SuperScript II reverse transcriptase (Invitrogen). When we used the modified protocol to reverse-transcribe and amplify 10, 1, 0.1, and 0.01 ng of human heart or liver total RNA (BD Biosciences Clontech), we obtained ample cDNA from one round of 23, 26, 29, and 32 PCR cycles, respectively (Fig. 1A). The addition of poly(dG/dC) facilitates the reverse transcription of longer transcripts as indicated by larger ranges of cDNA sizes and clearer band patterns, which are important for getting consistent good array data, but it does not adversely interfere with the reactions. Use of Superscript II instead of PowerScript in the reverse transcription step under otherwise similar conditions failed to yield visible amounts of cDNA in the agarose gel when starting total RNA was Յ10 ng, and a second round of PCR had to be performed to yield sufficient cDNA for synthesizing adequate amounts of cRNA for arrays. The number of genes detected on the arrays, however, was considerably lower than that obtained with PowerScript reverse transcriptase.The cDNA obtained with the modified protocol from 1, 10, and 100 ng of human total heart or liver RNA was in vitro-transcribed into complementary RNA (cRNA), which was then labeled with Bio-11-CTP and Bio-16-UTP (Enzo Biochem) by use of MAXIscript TM reagents (Ambion). The cRNA was then hybridized to the human GeneChip H133A (Affymetrix) as described previously (5 ). The numbers of genes detected as present (ϳ30%) were comparable among arrays prepared with various starting amounts of total RNA. We determined the reproducibility of the expression signals detected from these minute samples by running 2 replicate assays. Shown in Fig. 1B are the M-A plots of replicate arrays (M represent...

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Current awareness on comparative and functional genomics

2005

Comp Funct Genom

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Weed genomics: New tools to understand weed biology (Review). Trends Plant Sci 9: (8) 391. Baxevanis AD. 2003. Using genomic databases for sequence-based biological discovery (Review). Mol Med 9: (9-12) 185. Benning C. 2004. Genetic mutant screening by direct metabolite analysis (Review). Anal Biochem 332: (1) 1. Brouzes E, Farge E. 2004. Interplay of mechanical deformation and patterned gene expression in developing embryos. Curr Opin Genet Dev 14: (4) 367. Brunner AM, Nilsson O. 2004. Revisiting tree maturation and floral initiation in the poplar functional genomics era (Review). New Phytol 164: (1) 43.

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A method for gene expression analysis by oligonucleotide arrays from minute biological materials

Cited by 32 publications

References 25 publications

Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells

Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells

Amplification of Nanogram Amounts of Total RNA by the SMART-Based PCR Method for High-Density Oligonucleotide Microarrays

Current awareness on comparative and functional genomics

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