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RNA pre-amplification enables large-scale RT-qPCR gene-expression studies on limiting sample amounts

Joëlle Vermeulen1†, Stefaan Derveaux1†, Steve Lefever1, Els De Smet1, Katleen De Preter1, Nurten Yigit1, Anne De Paepe1, Filip Pattyn1, Frank Speleman1 and Jo Vandesompele1,2*

1 Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium

2 Biogazelle, Ghent, Belgium

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BMC Research Notes 2009, 2:235 doi:10.1186/1756-0500-2-235

Published: 25 November 2009

Additional files

Additional file 1:

RDML file 1. Primer sequences of the MYCN and MYCN regulated genes and raw data from the expression analyses on the 6 neuroblastoma cell lines.

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Additional file 2:

RDML file 2. Primer sequences of the MAQC target genes and raw data from the expression analyses on the 4 MAQC reference samples.

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Additional file 3:

Supplemental Material and Methods. Details on sample preparation, gene-expression analysis, formulas and raw data availability.

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Additional file 4:

Supplemental Figure S1. Frequency distribution (left-axis) and cumulative frequency (right-axis) of the difference in quantification cycle value (dCq) induced by sample pre-amplification (x-axis) for 194 genes measured in the 4 MAQC reference samples. There is a clear sequence-specific pre-amplification bias, meaning that some sequences or parts of transcripts pre-amplify better than others.

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Additional file 5:

Supplemental Figure S2. SPUD assay for the detection of enzymatic inhibitors in purified pre-amplified samples (P) and in non-purified pre-amplified samples (NP) with negative control (NC) and positives controls with known inhibitor (PC). Difference in Cq or delta-Cq (dCq) (NP or P vs. NC) < 1 indicates absence of enzymatic inhibitors.

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Additional file 6:

Supplemental Figure S3. PCR efficiencies estimated with two different single curve efficiency algorithms, PCR Miner (red) and LinReg (blue). Efficiencies of purified (squares; 2 replicates) and non-purified (triangles; 2 replicates) pre-amplified samples for each gene are comparable indicating that non-purified pre-amplified samples do not contain inhibitors and amplify with the same PCR efficiency.

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Additional file 7:

Supplemental Figure S4. Cumulative distribution plot of the delta-delta Cq (ddCq) before and after pre-amplification for 10 reference genes and 10 samples without purification of the pre-amplified product (black) and with purification of the pre-amplified product (grey). Each dot represents a ddCq-value between 2 samples before and after pre-amplification. Purification is not required for the preservation of differential expression.

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Additional file 8:

RDML file 3. Primer sequences of HPRT1 and SDHA and raw data from the expression analyses on 738 neuroblastoma tumour samples.

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