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Research ArticleArticle

Automated Extraction of DNA and RNA from a Single Formalin-Fixed Paraffin-Embedded Tissue Section for Analysis of Both Single-Nucleotide Polymorphisms and mRNA Expression

Guido Hennig, Mathias Gehrmann, Udo Stropp, Hiltrud Brauch, Peter Fritz, Michel Eichelbaum, Matthias Schwab, Werner Schroth
DOI: 10.1373/clinchem.2010.151233 Published November 2010
Guido Hennig
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Mathias Gehrmann
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Udo Stropp
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Hiltrud Brauch
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Peter Fritz
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Michel Eichelbaum
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Matthias Schwab
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Werner Schroth
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    Fig. 1. Schematic work flow of the automated DNA and RNA isolation procedure for FFPE tissue sections.
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    Fig. 2. Amplified genes and yields.

    (A), Absolute and relative number (%) of measured RT-qPCR results for 9 genes obtained from DNase I–treated eluate and qPCR results for the PAEP gene without prior reverse transcription from paired tumor (T) and adjacent normal (N) tissue block sections. (B), Plots of Cq values for the normalization gene RPL37A and the PAEP gene used as surrogate markers for mRNA or DNA yield, respectively. Cq values are shown for adjacent normal (N) or paired tumor (T) FFPE sections. Black bars indicate median Cq values of 26.3; 24.9; 32.2, and 31.1, respectively.

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    Fig. 3. Relationship between tissue cell count and RNA yield per tissue section.

    Cell numbers per defined tissue area were scored as 1–10, ranging between <50 cells [Harris et al. (1)] to >4000 cells [Bohmann et al. (10)]. Expression of the housekeeping gene RPL37A (RT-PCR Cq value) was used as a surrogate for overall RNA content per tissue section. The plot shows an inverse correlation between Cq values and cell numbers within a tissue section (Spearman r = −0.56), suggesting an unbiased tissue processing of the method with respect to RNA yield and downstream applications.

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    Fig. 4. Overview of quantitative mRNA expression.

    RPL37A-normalized quantitative expression values (DCq) of ESR1, PGR, HER2, EPHX1, BIRC5, MMP7, VEGFA, and TOP2A in FFPE sections from paired tumor (T) and adjacent normal (N) tissue block sections. Median Cq values are indicated by black bars and as numbers on top of the x axis.

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    Table 1. Investigated SNPs in tumor and normal breast tissue and the proportion of congruent genotypes.
    GeneChromosome locationrs No.Tumors genotyped, n (%)Normal tissues genotyped, n (%)Tumor-normal pairs, nIdentical/total chromosomes, n/N, (%)
    ESR16q25.1rs934079997 (92.3)101 (96.2)94187/188 (99.4)
    ESR214q23.2rs498693897 (92.3)100 (95.2)92183/184 (99.4)
    ESR214q23.2rs944052101 (96.2)102 (97.1)98194/196 (98.9)
    NCOA320q12rs223078298 (93.3)101 (96.2)95189/190 (99.5)
    NCOA320q12rs207654698 (93.3)101 (96.2)95188/190 (98.9)
    PPARGC1B5q32rs7732671102 (97.1)103 (98.1)100198/200 (99.0)
    RRAS211p15.2rs1102319796 (91.4)99 (94.3)91181/182 (99.4)
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    Table 2. MAF of investigated SNPs, test of HWE, and MAFs from a reference cohort.
    SNPTumor MAFHWE, PNormal tissue MAFHWE, PMAF referencea)Differences paraffin vs referenceb)
    rs93407990.2890.580.3120.690.349NS
    rs49869380.3970.950.4050.980.369NS
    rs9440520.1390.670.1370.990.154NS
    rs22307820.1070.990.1040.990.099NS
    rs20765460.0710.740.0690.770.098NS
    rs77326710.0590.510.0630.610.073NS
    rs110231970.3280.740.3330.990.379NS
    • ↵a Reference MAF refers to a cohort of breast cancer individuals (N = 920) genotyped from blood DNA (H. Brauch, P. Fritz, M. Eichelbaum, M. Schwab, and W. Schroth, unpublished data).

    • ↵b Two-sided χ2 test for differences in genotype frequencies between either tumor or normal paraffin tissue and the reference frequencies derived from blood DNA. NS indicates not significant.

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Clinical Chemistry: 56 (12)
Vol. 56, Issue 12
December 2010
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Automated Extraction of DNA and RNA from a Single Formalin-Fixed Paraffin-Embedded Tissue Section for Analysis of Both Single-Nucleotide Polymorphisms and mRNA Expression
Guido Hennig, Mathias Gehrmann, Udo Stropp, Hiltrud Brauch, Peter Fritz, Michel Eichelbaum, Matthias Schwab, Werner Schroth
Clinical Chemistry Dec 2010, 56 (12) 1845-1853; DOI: 10.1373/clinchem.2010.151233
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Automated Extraction of DNA and RNA from a Single Formalin-Fixed Paraffin-Embedded Tissue Section for Analysis of Both Single-Nucleotide Polymorphisms and mRNA Expression
Guido Hennig, Mathias Gehrmann, Udo Stropp, Hiltrud Brauch, Peter Fritz, Michel Eichelbaum, Matthias Schwab, Werner Schroth
Clinical Chemistry Dec 2010, 56 (12) 1845-1853; DOI: 10.1373/clinchem.2010.151233

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