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ORIGINAL ARTICLE

Correcting False Gene Expression Measurements From
Degraded RNA Using RTQ-PCR
Matthias Port, MD,*w Hans Ulrich Schmelz, MD,*z Tanja Stassen,y Kerstin Mu¨eller,*
Marcus Stockinger, MD,* Richard Obermair,* and Michael Abend, MD, PhD*

as cancer.1 The recovery of RNA from paraffin-embedded
Abstract: This paper describes a method allowing correcting tissue samples and the development of the polymerase
false gene expression measured on highly degraded RNA using chain reaction (PCR) method have significantly expanded
real-time quantitative reverse transcription-polymerase chain the opportunity for gene expression analysis not only of
reaction (RTQ-PCR). RNA was isolated from different models paraffin-embedded tissue samples but also of those that
(in vitro cell lines, in vivo models of human and dog) and are fresh and frozen.
different tissue types. In vitro RNA degradation and modeling It is widely known that RNA and DNA extracted
of in vivo degradation were applied on intact and degraded total from paraffin-embedded tissue blocks are of poor quality
RNA. Gene expression (eg, Bcl-2, GAPDH, PGK, PSME3, and degraded to various degrees compared with those
RAB2, BAX) was measured using RTQ-PCR. 18S rRNA from fresh and frozen tissues.2–6 Our own experiences
proved to be the most constant house-keeping gene. Less than with RNA isolated from paraffin-embedded tissue sam-
10-fold degraded RNA can be quantified correctly when using ples of different type (thyroid cancers, testis tumors and
18S rRNA for normalization purposes. Higher-fold degraded normal testis tissues, lymph nodes, kidney, liver) and
RNA can be quantified correctly up to a precision that is origin (human, dog, mice) always lacked in the successful
comparable to RTQ-PCR measurements on intact RNA when extraction of intact RNA, which is in line with the
simulating the RNA-species and tissue-specific degradation literature. Unfortunately, degradation of RNA can be
kinetic. also detected in frozen tissues sometimes caused from
Key Words: RNA degradation, RTQ-PCR, correction of false warm ischemia before extraction.7 However, when using
gene expression measurements, house-keeping genes, simulation state of the art technologies, such as immediate transfer of
of RNA degradation the fresh tissues excised during the operation into a
solution that inhibits RNases and stabilizes RNA-species
(Diagn Mol Pathol 2007;16:38–49) (eg, RNA-later, Qiagen), degraded RNA was usually
absent after RNA isolation.8
Nevertheless, the usability of RNA from archival

W ith the advent of molecular biologic methods, it
became desirable to apply these potent methods to
archival paraffin-embedded tissue samples. Isolation of
tissues has been reported for investigating gene expres-
sion.9–11 These and other groups reduced their research
on the detection of the presence or absence of a certain
RNA in particular seemed to be interesting, because RNA-species (yes or no answer). When considering the
expression alterations caused by gene abnormalities like well-accepted experience of RNA degradation taking
deletions, structural alterations, rearrangements, and place especially in paraffin-embedded tissues, the question
amplifications are associated with certain diseases such arises whether the lack in detection of a certain RNA-
species related, for example, to oncogenesis might be
caused by either degradation or the absence of this RNA-
From the *Bundeswehr Institute of Radiobiology, Munich; wDepart-
ment of Hematology and Oncology, Hannover Medical School; species. These considerations point to the need to develop
zDepartment of Urology, Federal Armed Forces Hospital, Koblenz; methods that allow correction of false gene expression
and yInstitute of Veterinary Pathology, Ludwig-Maximillian- measured on degraded RNA while taking into account
University, Munich, Germany. the degradation process.
Supported by the German Ministry of Defense. It is the purpose of the newly developed method
Competing interests statement: We declare that we have no significant
competing financial, professional, or personal interests that might described in this manuscript not only to focus on the
have influenced the performance or presentation of the work detection of certain RNA-species, but to quantify gene
described in this manuscript. expression after degradation occurred. With the method
Reprints: Hans Ulrich Schmelz, MD, Federal Armed Forces Institute shown herein, quantification on degraded RNA can be
of Radiobiology, Ernst-von-Bergmann-Kaserne, Neuherbergstr. 11,
D-80937 Munich, Germany (e-mail: michaelabend@bundeswehr.
performed with a precision resembling real-time quanti-
org). tative reverse transcription-polymerase chain reaction
Copyright r 2007 by Lippincott Williams & Wilkins (RTQ-PCR) measurement on intact RNA.

38 Diagn Mol Pathol  Volume 16, Number 1, March 2007

dilution in 0. Weitersheim. Braunschweig. Germany) according to the manufacturer’s the volumes of RNA-eluates of degraded RNA had to be instructions. Seifert. and the total RNA was isolated in general was diluted in water (1 mg/mL). The resulting cDNA trypsinized if necessary. Germany). MCF-7 cells received 10 mg insulin/mL of these biopsies were cut and weighed after being medium (Sigma. Germany). Qiagen. and colon. 1 volume of RNA-later solution and stored on ice before liver. DMEM medium (Sigma. metastasis. Aliquots of total RNA (1 mg) were reverse tran- Invitrogen. The dose rate was B1 Gy/min In vitro degradation was performed with RNase A at 13 mA. dest) and stored over 1 hour at room temperature. Number 1. Deisenhofen. protocols (Qiagen) was driven. age range: 30 to 82 y) were commercially available (which equals about 15 to 25 mL) were added to the same (Stratagene Europe. at pH 7. Deisenhofen. However. (TaqMan Gold RT-PCR Kit). Hilden. Exponentially growing cell cultures were equal to the volumes of intact RNA. ethanol-resistant RNase-cocktails isolated from the biop- kidney. RTQ-PCR Warrenton) and digested (proteinase K. Tissues were fixed in RNA-later enzymatic digestion of RNA-species. PeqLab. and spleen of 2 female dogs aged 1 and 15 years. MCF-7 and 28S/18S rRNA ribosomal band B2:1). Germany). nonseminomas (mixed nonseminomatous germ cell tu. and thermal cycled according to a 2-step PCR protocol remaining DNA digested (RNase free DNase Set. March 2007 Correcting False Gene Expression Measurements MATERIALS AND METHOD applying 7  106 cells per column using the RNeasy Mini Kit and DNA digestion as described above. In vivo degradation was performed with the Human RNA isolated from 3 organs. Ger- 5% CO2 in the air and hydrogen carbonate (Merck.Diagn Mol Pathol  Volume 16. 12). stored at r 2007 Lippincott Williams & Wilkins 39 .2  SSC (according to the guidance of the Tissue Samples and Histologic Examination manufacturer Roche) of stock RNase A (10 mg RNase A/ Testicular tumor biopsies from 37 patients (age 10 mL aqua dest) led to solutions containing 1 to 10  4 ng range 19 to 45) were analyzed. Deisenhofen. Amsterdam. 1 to 30 minutes.5-mL tube and carefully shaken. Hilden. A sequential 10-fold L929 cells with 6 Gy. scribed using Multiscribe reverse transcriptase and lated (RNeasy Mini Kit. squeezed manually to remove the liquid. Enzymatic reaction was run at 41C unaffected sites of the resected testis representing normal and 101C over 1 to 10 minutes to decrease the fast tissue were analyzed. Cells and Cell Culture Absorption of total RNA at A260/A280 (ratio had Human mammary carcinoma (MCF-7). Ahrensberg. Germany). ranged from 0. exponentially certain cases the solution was diluted 10-fold with aqua growing cell cultures were irradiated at room temperature dest to decrease the fast enzymatic digestion of RNA- with single doses of 240 kV x-rays (Isovolt 320/10. For reverse transcription. total RNA was iso. Culture condi. All human samples were obtained with by the manufacturer (Qiagen). many) containing 1 mL of 50% ethanol (diluted in aqua Darmstadt. TNM classification RNase A/mL. informed consent. After Radiation Conditions removal of the tissue. Karlsruhe. Mannheim. In addition. RNA Isolation and Quality Control Tissue was homogenized (homogenizer. Biopsies were stored in RNA-later solution. About 25 mg tions were 371C in a humidified atmosphere buffered by was given into a tube (Eppendorf. L929) or in cell suspension tiation between degraded (smear) and intact RNA (ratio (HL-60) and subcultured twice per week. Omni. 20 mg/mL. 29 pure seminomas and 8 were filled into a 0. Qiagen.4. species. the RNA-isolation cleanup according to conventional Tissues were immediately fixed in RNA-later solution. of 9 different individuals (4 females. this solution was immediately used for RNA degradation purposes or stored at  201C. The absorbed dose was measured with a Duplex dosimeter RNA Degradation (PTW. In Twenty-four hours after passaging. Germany) were grown either as a Germany). skin. MCF-7 cells were irradiated with 2 and 6 Gy and (Sigma Deisenhofen. The total mors) were examined.2  SSC and 10 mL of the precooled RNase-solutions T1N0M0 to T3 N2 M1. Heidelberg. Erlangen. Hilden. a certain volume of precooled (tumor.5-mL tube 15 to 25 mg of RNA males. Pieces Additionally. 20 mg of precooled RNA were added. The reaction was stopped by adding Tissue samples were taken from lymph nodes. node. see Ref. Germany). Germany). Germany) filtered with 3 mm Be. Germany) and L929 cells in (40 cycles using b-actin primers). Netherlands and BD volume of the RNase-cocktail and incubated at 301C over Bioscience. Germany). Typically. Agarose gel electrophoresis allowed differen- monolayer culture (MCF-7. and human leukemia (HL-60) cells spectral photometry (Nano Drop. Freiburg. Media were supplemented with a 10% heat-inactivated fetal calf Release of Tissue-specific RNases serum (Boehringer Mannheim. mouse to be >1. Germany). Typically. 37 biopsies of volume was 50 mL. Reaction was solution (Qiagen. Germany). DNA contamina- HL-60 cells were maintained in RPMI 1640 medium tion of the probes was checked with conventional PCR (Sigma.8). Germany) immediately after stopped by adding RLT buffer and running the conven- surgery. and quantification was examined with fibroblast (L929). some of these tissues contained tional RNA-isolation cleanup protocol as recommended degraded RNA. in a 0. Finally. 5 sies. (DSMZ. namely liver. Spectral photometric quantification of RNA followed the next day.

1). and 10 ng cDNA. and PGK) were commer. 2B).5 (Fig. which uses the 50 nuclease activity 10-fold differences in absolute 18S rRNA gene expression of Taq DNA polymerase to cleave a TaqMan probe could be found. cell. so-called threshold-cycles (CT) of the PCR-reaction of Examination of absolute 18S rRNA gene expression unknown samples into ng cDNA.Port et al Diagn Mol Pathol  Volume 16. RNase-free water was added to a final volume of 30 mL per reaction. Seven primers and probes (GAPDH. which corresponds to a PCR efficiency 6 Gy) and certain time points after irradiation (up to 72 h) >90%). 1 cially available as so-called assays on demand. with higher degree of degraded RNA the Kidney Kidney Kidney Colon Colon Colon Liver Liver Liver amount of template had to be increased from 10 ng of cDNA up to 500 ng of cDNA. thus allowing conversion of the higher compared with PGK and HPRT. Unirradiated (control) values of 18S rRNA were in the same order of magnitude Statistics compared with the irradiated probes. with annealing house-keeping genes in 3 tissue types of 9 human individuals. within each model system (Fig. L929). absolute gene Plot 2000. linearity lasted over 6 log units.0001 However.13 The PCR reaction typically included 0. Erkrath. In contrast. All materials used for RTQ-PCR were human RNAs isolated from liver. 40 r 2007 Lippincott Williams & Wilkins . Germany). absence of inhibitory substances in the RNA probes. and species- independent height of differential 18S rRNA gene RESULTS expression with a variance of 0. dog).1 primers and probes (PSME3 and PKCI) were used as described before. all of which revealed lower The means. March 2007  201C. for example. in some cases values compensated model-specific differences in absolute due to restricted material these conditions had to be gene expression and demonstrated an almost homoge- changed which is mentioned in the legend.01 0. employing RTQ-PCR. Jandel. The same pattern The gene expression analysis of genes in general was could be found for testis tumors and their corresponding done in triplicate and duplicate. The dynamic range of in different species (mouse. Certain RNAs (marked with an asterix in Fig. A relative standard curve derived from sequential 8-fold dilutions of stock cDNA of known quantity (lasting from 0. A hot-start Taq. Absolute comparable efficiencies (93% to 96%) of the PCR- gene expression of both genes typically differed 100% reaction evolved (Fig. n = 3. Number 1. 9 individuals or dog tissues. HPRT. When <0. Weiterstadt. However. Aliquots of these RNAs were 3 human tissue types of 9 individuals revealed mean taken and diluted to search for substances present in the differences for absolute 18S rRNA gene expression of probes which might inhibit the PCR-reaction.4 to 3.001 FAM labeled probe. 300 nM forward and 300 nM reverse primer. and used as a template for subsequent PCR 10 reactions. Every experiment expression of 18S rRNA relative to appropriate control was performed at least 3 times. Rab2. 1). PCNA. kidney. time.5 ng cDNA until 7. 2A). neous and radiation dose. colon). Moreover. and colon of ordered from Applied Biosystems. and elongation of primers and probes occurring at 601C Bars represent the mean calculated from 3 individuals.6 fg cDNA per reaction) was used for a linear regression analysis absolute gene expression of 18S rRNA was 2 log scales of unknown samples. Therefore. tissue. SEM. organ. among L929 cells and during PCR. in curves was almost constant throughout the experiments vitro cell lines (MCF-7. Comparison of absolute gene expression of 3 minutes. 0. Germany. human.2-fold (20%) among the 3 organs and among the plotting absolute gene expression of 18S rRNA equiva- same organs of 3 individuals (Fig. radiation doses (2 and (range 3. organs (liver.6. a linear specific mean differences in absolute gene expression were regression with almost similar slopes (3. The slope of the standard kidney. Forty PCR cycles were driven. 2C). Two self-designed 0. up to Version 1. Error over 1 minute followed by a denaturation step at 951C bars show the SEM. Analysis of gene expression was generated using showed almost comparable values for gene expression a GeneAmp 5700 Sequence Detection System (SDS. One (relative to 18S in liver) predeveloped assay for detection of 18S rRNA (used for RNA-equivalents normalization purposes) was used. 200 nM 0.5 volume of the 2  concentrated TaqMan Master Mix containing hot start AmpliTaqGold DNA polymerase.5) and 10-fold and 2-fold for PGK and HPRT (Fig. HPRT PGK 18S Polymerase was used and activated at 951C over 10 FIGURE 1.4 to 3. In contrast. testis tumors and normal testis tissues. TaqMan). CDK4. This was indicative for the among the same organs of 3 individuals. over 1 minute. 2B) House-keeping Genes showed up to 15-fold differences in absolute gene Comparison of 3 common house-keeping genes in expression of 18S rRNA.3. normal tissues (controls). and minimum and maximum values absolute 18S rRNA gene expression compared with dog were visualized with the aid of graphical software (Sigma or other human organ tissues. Bcl-2. lents versus CT-values for all 4 diluted probes.

a differential gene expression relative In Vitro Degradation Kinetic and Normalization to the intact RNA-species (degree of degradation Degradation of human MCF-7 RNA with RNase A equals 1) was calculated (Fig. Depicted in vitro values reveal a representative selection of 12 further experiments not shown herein. This ratio was called ‘‘differential gene expression. 2C). This was comparable with comparable with undigested RNA. Three cDNA-probes (marked with an asterix in Fig. However. thus leading to a degree of degraded RNA. 1B) were diluted. middle and lower panel). right panels). After normalizing the 3 genes versus 18S rRNA. The abundance of the gene of interest under certain conditions relative to the abundance of the same gene under control conditions was built.Diagn Mol Pathol  Volume 16. 2C. PSME3 and PKCI revealed only a decrease of their which had no influence on the differential gene expression absolute gene expression over 2 log scales (Fig.52 L929. 3. 3. left as shown in Figure 2B. slope: -3. which showed a similar decrease degradation degree of 6 (one-sixth of intact molecules are r 2007 Lippincott Williams & Wilkins 41 . 3. slope: -3. Gel electrophoresis revealed degraded RNA in the (Fig.37 Nonsem: normal Nonsem: tumor 30 control RNA CT MCF-7 L929 25 20 15 1e-6 1e-5 1e-4 1e-3 1e-2 1e-1 1e+0 1e+1 Absolute gene expression (18S RNA-equivalents) FIGURE 2.43 Standard. left upper panel). Differential reduced absolute gene expression of 18S rRNA over gene expression increased with increasing degree of almost 5 log scales. gray columns.43 35 MCF7. March 2007 Correcting False Gene Expression Measurements A B C 40 Nonseminom. slope: -3. white lower degraded RNA showed differential gene expression columns in the left panels). A. nonseminoma normal and tumor tissue (Fig. 3. Measurements were performed in different models as outlined in the graphs. Gel electrophoresis revealed degraded testis tissue (inset in Fig. B. inset). As a tendency it could be shown that degradation covering nearly 5 log scales (Fig. Slope values for the selected 4 probes are depicted in the corresponding legend. Number 1. and 18S RNA equivalents measured.’’ C. For instance. Absolute gene expression of 18S rRNA was measured for each PCR-reaction containing 10 ng RNA equivalents. slope: -3. at a GAPDH degradation.

Aliquots of these solutions presumably 1e-4 containing RNases were transferred in tubes to digest 1e-5 intact human RNA of other origin (HL-60 cells). 3.6. expression appeared over 2 log scales for Bcl-2. present. 1e+0 3 PSME3. 1e-3 4 60. lower graph. 4.7 for GAPDH. lower 1e+0 graph) so that the plots converged against 18S rRNA Differential gene expression (relative to undigested RNA-species) 3 1e-1 with higher degraded RNA. With further increase in PCNA. and black circles). although spectral photometry measure- 1e-2 ments suggested a decrease in RNA amount taking place 1e-3 at 120 minutes.2 6 169 615 1e5 1 1. After normalization kinetics of different genes were always linear. These degradation kinetics 1e-2 2 allowed us to calculate an almost correct differential gene 1e-3 expression for the 3 genes at a degree of degradation Absolute gene expression (RNA equivalent) 1e-4 1 equal to 10 (one-tenth of intact molecules are present. lower inset. inset (RNA-equivalent) 1e-1 upper graph). 1e-7 This was associated by a decrease in the amount of RNA 1e+0 1e+1 1e+2 1e+3 1e+4 1e+5 x-fold degraded RNA (relative to undigested 18SrRNA) (Fig.00. Absolute gene expression PSME3 white rectangles. Number 1.9 for GAPDH. The lower graph reflects the degradation A piece of not degraded testis biopsies stored in kinetics of the 4 RNA-species examined. which was calculated relative to incubated in aliquots of the 50% ethanol solution over absolute gene expression of undigested 18S rRNA. The 1e-6 28/18 S bands disappeared after a 10-minute incubation. and PKCI. 4. which equals 17%). PSME3  0. Moreover. lower graph). and PCNA. several times. expression of degraded RNA-species was plotted versus the The RNA isolated from the remaining tumor was x-fold degraded RNA-species. and PKCI. the differential gene expres.04. 4. Concomitant the amount of RNA decreased (Fig. which became more 1 1.Port et al Diagn Mol Pathol  Volume 16. and white rectangles). CDK4. 1e-5 which equals 10%). Rab2. lower (gray columns) and 18S rRNA (white column). respectively. Fig. The absolute gene expression of 3 genes (Bcl-2. The 18S rRNA normalized differ- 1e-6 0 1e+1 4 ential gene expression relative to the corresponding PSME3 PSME3 undigested values was 1. Isolated RNA from these pieces 1e-4 2 run in an agarose gel showed a disappearance of the 28/ 1e-5 0 18S bands while a smear appeared. and 120 minutes. 5. Absolute gene RNA-later was taken and transferred into 50% ethanol. March 2007 Raw data 18S normalization (slopes: GAPDH  0. PKCI  0. The in vitro degradation found for CDK4. the known 1e-1 RNA-specific degradation kinetic represented by appro- 1e-2 2 priate regression models allowed to extrapolate from the 1e-3 1 degraded RNAs to the correct intact RNA expression 1e-4 values (Fig. This 1e+2 degradation process could be avoided when diluting 18 S rRNA 1e+1 GAPDH ethanol not in water but in RNA-later solution (Fig. but the versus 18S rRNA for 3 genes differential gene expression slopes of the 3 genes regression curves were lower similar to undigested RNA-species was found up to a 42 r 2007 Lippincott Williams & Wilkins . upper graph). 1e-5 0 1e+1 14 PKCI PKCI In Vivo Degradation 1e+0 12 10 Release of Tissue-specific Ethanol Resistant RNases 1e-1 8 Pieces of the same tumor biopsy were incubated in 1e-2 6 an ethanol 50% solution (diluted in aqua dest) over 30. PCNA) and 18S rRNA showed a reduction over almost 4 log scales for 18S rRNA (Fig.2 6 169 615 1e5 x-fold degraded RNA prominent with prolonged incubation time of the tissues (relative to undigested 18SrRNA) in the alcohol solution. 3.85. upper graph).2. whereas the graph. The decrease in absolute gene sion measured was 1. 4. black circles. 1. Rab2. upper inset. and 1. and 1. Rab2. and PSME3. right lower panel). Moreover with 106-fold to 676-fold degradation was up to 12-fold (PKCI) over undigested conditions no further reduction in absolute gene expression was (Fig. respectively. 4. whereas CDK4 showed a decrease over about 1 degraded RNA differential gene expression of the genes log scale. No degradation of HL-60 RNA was found after incubation FIGURE 3. 90. After normalization (18S rRNA). human RNA was in vitro degraded using RNase A. differential gene expression was Degradation Kinetic calculated relative to the undigested RNA-species (equals 1-fold degradation). Up to 120 minutes intact 1e+0 PKCI 28/18S bands and no smears were visible (Fig.65) 1e+1 4 GAPDH GAPDH compared with 18S rRNA (slope:  1.93. upper graphs). left panel. 1. The left panels of the upper graph depict the individual decrease in absolute gene expression of 3 genes in an RNase-free ethanol 50% solution (Fig.00. 3.

Intact human RNA was Rab2 PCNA incubated into a 50% ethanol-solution (diluted in aqua 1e-5 bidest). 1:1 1e-3 3 Absolute gene expression (RNA equivalent) 0.4 1e-1 25 CDK4 20 CDK4 Differential gene expression (relative to undigested RNA-speicies) 4 1e-2 Et OH/aqua bidest. lower graph). 5. Error equations of second or third order (Fig. upper graph). which previously contained a piece of tumor tissue. 1 10 100 1000 The degradation of the RNA increased with incubation time x-fold degraded RNA (relative to undigested 18SrRNA) (line 1 to 9 upper inset lower graph). These 18SrRNA 1e-4 representative experiments were repeated more than 3 times Bcl-2 CDK4 and performed on other tissues. The left panels of the upper graph depict the not be observed after incubation of RNA in a clean 50% individual decrease in absolute gene expression of 3 genes ethanol-solution. 1 2 3 4 5 6 7 8 9 PCNA 4 PCNA 8 1e-2 3 1e-3 6 2 0 1 2 3 4 5 10 15 1e-4 Incubation time (min) 1 1e-5 FIGURE 4. The lower graph reflects the degradation kinetics of being 3 times over values of intact RNA.Diagn Mol Pathol  Volume 16.6 1e-4 4 1e-5 2 1e-6 0 0. (gray columns) and 18S rRNA (white column). March 2007 Correcting False Gene Expression Measurements Raw data 18S normalization Std. 1 2 3 4 5 6 7 8 9 Rab2 4 Rab2 1e-2 3 14 1e-3 RNA (µg) 2 1e-4 12 1e-5 1 10 1e-6 0 1e-1 Std. Number 1. 1 2 3 4 5 6 7 1e-1 0. upper 1e-1 graph). whereas the human testis-RNA was degraded using an ethanol-solution previously containing a piece of testis tumor. Neither smear (line 2 and 3 inset upper graph) nor Absolute gene expression (RNA equivalents) decreased amounts of RNA were found when diluting ethanol 1e-2 in RNA-later solution (white rectangles. x-fold degraded RNA (relative to undigested 18SrRNA) phoresis. After normal- 13-fold degraded RNA (Fig. CDK4 showed a differential gene expression dation). Absolute gene expression of degraded RNA in most cases showed a several-fold degraded RNA-species was plotted versus the x-fold degraded higher differential gene expression compared with the RNA-species. right panels upper graph). line 4 to 7 inset upper graph). r 2007 Lippincott Williams & Wilkins 43 . expression of undigested 18S rRNA. This degradation could FIGURE 5.2 Et OH/RNA-later. How- ever. 1:1 1e-4 2 0 20 40 60 80 100 120 1e-5 1 18 1e-6 0 1e-1 16 Std. ization (18S rRNA) differential gene expression was calculated relative to the undigested RNA-species (equals 1-fold degra- However. at 120 minutes spectral photometry measurements 1e-3 suggested a decrease in RNA amount. bars represent the SEM. These representative tion kinetics of the 4 RNA-species was characterized by results were performed in duplicate at 3 different tissues. RNA isolated from pieces of tumor biopsies that 1e-6 0 were previously incubated in 50% ethanol (diluted in aqua 1 3 13 106 676 1 3 13 106 676 bidest) were degraded versus time (shown by gel electro. Greater 13-fold the 4 RNA-species examined. which was calculated relative to absolute gene undigested RNA-species measured. Symbols represent mean values of measurements done in triplicate. This was associated with a decrease in the amount of RNA (black circles. 5. The in vivo degrada.8 Bcl-2 10 Bcl-2 1e-2 18SrRNA 8 µg RNA/ mg tissue 1e-3 6 0.

For instance. While Bcl-2 and CDK4 degradation appeared a biopsy. these degradation kinetics appeared depen- correct values of intact RNA with the aid of the dent on the biopsy/ethanol solution. However. after digestion of 3 other tissues but in the which previously contained a piece of the same tissue same ethanol solution that previously contained a piece of biopsy. individuals into their corresponding ethanol solutions. The measurements shown in A were performed on intact RNA of 3 other tumors. 6A). Intact RNA was isolated from 3 tumors and partly digested in ethanol-solutions that previously contained the corresponding tumor tissues. Absolute gene expression of 4 genes was measured in RNA-probes characterized by increased degradation. A. B. Number 1. These representative results were performed as single measurements and using 9 different tissues. the individual plots of the biopsies discovered A 1e-1 Bcl-2 Rab2 1e-2 1e-3 1e-4 Absolute gene expression (RNA equivalents) 1e-5 1e-6 1e-7 1e-1 CDK4 PCNA 1e-2 1e-3 1e-4 1e-5 biopsy 1 1e-6 biopsy 2 biopsy 3 1e-7 1e+0 1e+1 1e+2 1e+3 1e+4 1e+0 1e+1 1e+2 1e+3 1e+4 B 1e-1 Bcl-2 Rab2 1e-2 1e-3 Absolute gene expression (RNA equivalents) 1e-4 1e-5 1e-6 1e-7 1e-1 CDK4 PCNA 1e-2 1e-3 1e-4 1e-5 biopsy 4 1e-6 biopsy 5 biopsy 6 1e-7 1e+0 1e+1 1e+2 1e+3 1e+0 1e+1 1e+2 1e+3 x-fold degraded RNA (relative to undigested 18S rRNA) FIGURE 6. The known RNA-specific degradation kinetic istic could be shown for Rab2 and PCNA (Fig. lower graph). but these RNAs were partly digested in the same ethanol-solution that contained a tumor tissue previously. a linear quadratic degradation character- different. March 2007 The kinetic of every RNA-species appeared individually almost linear. regression curves (Fig. 5. allowed to extrapolate from the degraded RNAs to the Additionally. 44 r 2007 Lippincott Williams & Wilkins .Port et al Diagn Mol Pathol  Volume 16. depending on the biopsy absolute gene expression of Degradation kinetic of the 4 RNA-species was CDK4 showed a more than 15-fold difference at a examined after incubating the isolated RNA of 3 1000-fold degradation.

For 27 measurements the same fold-18S rRNA-degradation. CCND2. Absolute gene expression of PCNA. and Bax was measured in duplicate in 3 patients. Intact RNA was isolated from tumor tissue and the corresponding normal tissue.1 1 10 100 1000 10000 1e-7 x-fold degraded RNA 1 10 100 1000 10000 x-fold degraded RNA FIGURE 7. CCND2. When using the method as described above. dashed gray line (marker for intact RNA. Then the predicted gene expression values of degraded RNAs of the same tumor were used to calculate differential gene expression. RNases released by incubation of a piece of the tumor differential gene expression on degraded tumor RNA biopsy in ethanol (see above). In parallel. gray vertical arrow). 1. For instance. 6B). Then the regression curves allowed the reading of predicted absolute gene expression values before the degradation of RNA occurred (exemplified in left upper graph with curved arrow). and on intact normal tissues (controls) could be calculated. For the RNA isolated from HL-60 cells. at a 1000-fold degradation the tumor biopsies (Fig 7. 7. and Bax relative to the corresponding (to calculate differential gene expression). were digested up to 3000-fold in the tumor-specific respectively. Differential gene expression of intact RNA isolated from not degraded tumor tissues relative to normal tissue was performed. Intact RNA and degraded could be calculated with the help of the regression lines. On the not Bax) were measured in not degraded tumor biopsies degraded testis biopsies the differential gene expression of (3 individuals). lower right graph). CCND2.Diagn Mol Pathol  Volume 16. Number 1. and 3. 6B). which in the graph equals the value 1) represented the recalculated Precision of the Method gene expression before the process of degradation Gene expression of 3 genes (PCNA. March 2007 Correcting False Gene Expression Measurements before converged to 1 plot in all 4 RNA-species examined curve was moved into the data points of the degraded (Fig. The differential gene expression of intact tumor RNA was then compared with the differential gene expression measured on degraded tumor RNA of the same tumor using the method as described above.6. Under consideration of indicative for a similar result. this calibration (3 genes measured in 3 individuals and using 3 stages of 1e-1 PCNA CCND2 1e-2 1e-3 1e-4 Absolute gene expression (RNA equivalents) 1e-5 1e-6 normal tissue tumor tissue HL-60 degraded 1e-7 1 10 100 1000 10000 1 10 100 1000 10000 1e-1 Bax of undegraded /degraded probes 1e-2 9 differential gene expression 1e-3 7 5 1e-4 3 1e-5 1 1e-6 0. triangles) and plotted versus the performed (Fig. Aliquots of intact tumor RNA were also digested in RNase-cocktails released from individual pieces of tumor tissues incubated in ethanol-solutions. The results of 27 measurements (3 stages of degradation measured for 3 genes in 3 patients) are depicted in the lower right graph. intact HL-60 RNA was also digested in these RNase-cocktails. CCND2. The intersec- difference in CDK4 absolute gene expression between the tion where the regression line crossed the vertical long 3 biopsies was negligible (Fig. A ratio of 1 was degree of 18S rRNA degradation. Representative results from 1 patient are shown. Then aliquots of intact biopsies shown in Figure 7 differential gene expression RNA isolated from these tumor biopsies and HL-60 cells were 0. gene expression values of the 3 genes measured at RNA Then the ratio between the known (undigested biopsies) isolated from HL-60 were fitted with an appropriate and the recalculated differential gene expression was regression curve (Fig. Dashed lines represent the 99% confidence level. 7. and occurred and could be read from the y-axis.9 for PCNA. their corresponding normal tissues PCNA. and Bax. Regression curves of HL-60 RNA were moved into the data points measured for degraded tumor tissues (exemplified in left upper graph with vertical arrow). r 2007 Lippincott Williams & Wilkins 45 .9.

In parallel. A intact normal tissue RNA tumor-RNA 6 7 26 97 158 x-folded graded RNA B C PCNA FasL CLU CCND2 Bax 100 Differential gene expression (relative to normal tissue) 10 1 0. 46 r 2007 Lippincott Williams & Wilkins . The degree of 18S rRNA degradation is depicted in (A).000-fold in the tumor-specific RNase-cocktails released from tumor biopsies. and these values were taken for calculation of differential gene expression. On the basis of these calibration curves the absolute gene expression before degradation of RNA occurred was calculated (B).01 Seminoma Non-Seminoma degraded RNA degraded RNA degraded RNA degraded RNA degraded RNA intact RNA intact RNA intact RNA intact RNA intact RNA FIGURE 8.2 with an SD of 0. Five genes (PCNA. (Fig. in which gene expression was measured in the testis biopsies. Clusterin. Gene expression of genes was plotted versus degree of 18S rRNA degradation of HL-60 RNA and connected with a regression curve (B. RNA was isolated from 5 degraded testis tumor biopsies and their corresponding intact normal tissues (A). Number 1. Furthermore. Biopsies up-regulated or down-regulated genes always appeared RNA was isolated from 5 degraded testis tumor similarly up-regulated or down-regulated in the degraded biopsies and their corresponding intact normal tissues RNAs after recalculation as described above. Aliquots of RNA isolated from HL-60 cells were digested up to 10.Port et al Diagn Mol Pathol  Volume 16. are shown (B). Fas-Ligand. Four of the 5 genes. black line). 8A).7. differential gene expression was also calculated on a second cohort of 20 testis tumors and normal tissues with intact RNA (C).1 0. it could be shown that the geometric Application of the Method on Degraded mean ratio was 1. March 2007 RNA degradation).

right panels of upper graph). The total RNAs of 3 shown). over 6 log scales. It could be shown that the 18S Often total RNA isolated from biopsies not stored rRNA gene expression in opposite to other genes (PGK. lower graph). ethanol-resistant RNases were released between different cell lines or tissue types (Fig. However. 3. lower graph). As a result. 4. Degradation kinetic in this model for but increased with further degradation of RNA (Fig. when correcting absolute graphs). 4. a corrected expression of RNA-species could be plotted versus degree of 18S rRNA degradation of HL-60 estimated (Fig.14 Besides the fact ized by intact RNA. Therefore. The degradation kinetic was characterized equation. cially available RNase A allowed to examine the differential gene expression of the RNA-species was degradation kinetic among different RNA-species more comparable to the control values (intact RNA-species). 2B). 8C). differential gene degradation. 3. these tissues were incubated into the appropriate solu- An in vitro degradation model using the commer.000-fold in the tumor-specific RNases released RNA-species). demonstrated that depending on the amplicon size the detected amount of 18S rRNA decreased in parallel with almost similar slopes (data not shown). 3. 5 each RNA-species was characterized by a first order upper graphs). 5. control probes and the altered of Perlmutter et al. differential gene expression was also RNA-species and increased with the height of degraded calculated on a second cohort of 20 testis tumors and total RNA (data not shown). tions. (Fig. with an up to a 10-fold degradation. but tissue material is generally Method).15 Their enzyme activity could be probes showed similar 18S rRNA abundance so that inhibited by dilution of ethanol in RNA-later (Fig. 8B reveals (Fig. this approach normal tissues with intact RNA. when knowing the height of by incubation of a piece of the tumor biopsy in ethanol degradation and the RNA-species’ individual degradation (see above). When we searched for the reason. differential gene expression (relative to the adequate upper graph). upper graph).Diagn Mol Pathol  Volume 16. lower (Fig. in RNA-later solution appeared degraded (own experi- HPRT) appeared almost constant within a certain cell/ ence). fragment of degraded RNA-species. we recently detected (Fig. Hence. However. Active ethanol resistant RNases could be control values) between different models appeared com. Number 1. thus suggesting that released RNases become present in the isolated total RNAs and characteristic of denatured with higher concentrated ethanol (data not the cell and tissue types examined. With this in regressions corresponding to PCR efficiencies of 93% to mind ethanol-resistant RNases were released from pieces 96% were indicative for the absence of differences in the of testis tumors or normal testis tissues. Gene expression of the same 5 genes was kinetic. only isolated when using 50% to 70% ethanol. at higher parable (Fig. 3. 10-fold degradation differential gene expression of the Aliquots of RNA isolated from HL-60 cells were digested RNA-species was comparable to the control values (intact up to 10. March 2007 Correcting False Gene Expression Measurements CCND2. carefully (Fig. In parallel. it became tissue type provided an equal amount of the template obvious that during the conventional dehydration steps of cDNA was given into the PCR-reaction (Fig. In the first step. These RNase-cocktails could also be used to degrade The linear regressions and the equal slopes of all RNA of other origin (Fig. 4. 2A). When comparing the seemed inappropriate. This suggested r 2007 Lippincott Williams & Wilkins 47 . no RNA degradation could be gene expression could be caused by inhibitory substances found. The differences in 18S rRNA absolute ethanol concentration. When calculating DISCUSSION differential gene expression relative to control values gene To correct false gene expression measurements in expression of both probes will become altered in the same degraded tumors. lower graph). 6A). up to a representative results of 4 genes) in the testis biopsies. and Bax) were measured (Fig. To develop the degrada- RNA and connected with a regression curve (Fig. this might also explain the results Within these models. 2C). 8B. tissues of different origin. 3). in previous experiments we the tumors before the degradation of RNA occurred showed that amplification rate was dependent on the (Fig. Recently. Commercially available kits allow amplification were taken to estimate the absolute gene expression in of total RNA. Even in vivo. Therefore. no significant differences could be that it will become difficult to manage this. Corresponding amount of inhibitory substances among these RNA intact total RNAs isolated from the remaining pieces of probes (Fig. large amounts of RNA isolated from tissues black line). this biopsy and gene-specific regression curves limited. RNA-species digested in their corresponding gene expression of the RNA-species by 18S rRNA RNase-cocktails released from the same tissue showed absolute gene expression. we searched for an adequate expression will be equal although the absolute amount house-keeping gene with almost constant copy number in of RNA-species would be smaller in the probes. 18S the tissue preparation taking place before paraffin rRNA absolute gene expression differed up to 15-fold embedding. Furthermore. it is necessary to quantify the height of order of magnitude. differential gene expression of regressions characteristic for the RNA-species and the RNA-species increased with the height of degradation differed between the tissues (Fig. but the slopes differed among the RNA-species by equations of second or third order and more complex and were slightly smaller compared with 18S rRNA compared with the in vitro experiments (Figs. tion kinetic. it was suggested that the recalculated differential gene expression of the degraded detection system used must fit the length of the RNA- tumor tissues with another cohort of tumors character. 1). As shown above (section Precision of the would be necessary. 8B). it might be advisable to perform the cell and tissue types and standard total RNA were diluted dehydration procedure with >70% ethanol solutions. and the PCR efficiency was calculated.

For a degree of degradation smaller than 10. These calibration curves were used to estimated (Figs. normalization with 18S is sufficient. 5. an in vitro simulation of the degradation leads to a precision that is comparable to RTQ-PCR measurements on intact RNA. estimate the precision of a method for recalculation of 48 r 2007 Lippincott Williams & Wilkins .Port et al Diagn Mol Pathol  Volume 16. move calibration curve into data points of degraded RNA species gene expression: extrapolation of degraded target gene to degradion value of 1 = target gene in intact RNA FIGURE 9. March 2007 measurement of 18S gene expression in any given intact probe of the same tissue type measurement of 18S gene expression in degraded and intact probe calculation of the degree of degradation (18S degraded RNA/ 18S intact RNA) yes no degree of degradation <10 measurement of target gene in measurement of target gene in degraded probe degraded probe eluation of tissue specific ethanol normalization with 18 S resistant RNAses and degradation of intact reference RNA gene expression: measurement of target gene and 18 S degraded target gene = in intact and artificial degraded target gene in intact RNA reference RNA for calibration curve degraded gene expression values and calibration curve values are plotted vs degree of 18S rRNA degradation. for a higher degree of degradation. (Fig. that the released RNase-cocktails were tissue specific. Number 1. 6B). 6). corrected create biopsy (or RNase-cocktail) and gene-specific gene expression on degraded RNA-species could be calibration curves. intact human HL-60 RNA (which can be With the knowledge of the height of degradation and the isolated in the desired amounts in vitro) was used to RNA-species’ individual degradation kinetic. Because RNA from different origin becomes de- These differences vanished when digesting RNA from graded similarly when using the same RNase-cocktail different tissues but in the same RNase-cocktail (Fig. 6B). Schematic representation of the technique applied to correct false gene expression measurements from degraded RNA using RTQ-PCR.

8). Pizzocaro G. paraffin-embedded liver tissue. Diagn Mol Pathol.39:317–361. ing to the proposed method the calculated differential 13. Shieh YS. the calculated geometric mean was closed to the 4. Diagn Mol tion of 1 in Fig. Kiviat NB. With an SD of 0. Pathol. Comparison of snap In summary. et al. J Mol Diagn. Specht K.17 meaningful results could be measured in degraded 1994. After calculation of the 16. J Clin Oncol. 2001. Int J Radiat Biol. with higher 2005. Moutschen-Dahmen M. paraffin-embedded tissues by PCR. r 2007 Lippincott Williams & Wilkins 49 . et al. formalin-fixed. 1991. Reesink HW. 1991. Port M. 2001. Am J Pathol. 1977. Stockinger M. thus allowing us to calculate differential gene expression REFERENCES before degradation occurred (depicted as x-fold degrada. Goldstein DR. Mutat expected value (1. Guha-Thakurta N. in case of degraded RNA >factor 10 accord. 8C).48:267–272. 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