Clinical Chemistry AACC Online Job Center
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 53: 587-593, 2007. First published February 8, 2007; 10.1373/clinchem.2006.078592
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
clinchem.2006.078592v1
53/4/587    most recent
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Langebrake, C.
Right arrow Articles by Reinhardt, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Langebrake, C.
Right arrow Articles by Reinhardt, D.
Related Collections
Right arrow Molecular Diagnostics and Genetics
(Clinical Chemistry. 2007;53:587-593.)
© 2007 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Preanalytical mRNA Stabilization of Whole Bone Marrow Samples

Claudia Langebrake1,a, Kalle Günther2, Jürgen Lauber2 and Dirk Reinhardt1

1 Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany.
2 Qiagen GmbH, Hilden, Germany.

aAddress correspondence to this author at: Medizinische Hochschule Hannover, Department of Pediatric Hematology and Oncology, AML-BFM Study, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. Fax 49-511-532-9029; e-mail: claudia{at}langebrake.de.


   Abstract
Top
 Abstract
Introduction
 Patients, Materials, and Methods
 Results
 Discussion
 References
 
Background: Gene expression profiling is a useful tool for cancer diagnosis and basic research. A major limitation is that, even during short-term storage of native specimens of peripheral blood or bone marrow (BM) and/or RNA isolation, significant changes of gene expression pattern can occur because of gene induction, repression, and RNA degradation.

Methods: We investigated the effectiveness of a newly developed RNA stabilization and preparation system for BM specimens (PAXgeneTM Bone Marrow RNA System) over time. We analyzed 256 RNA samples, processed from 64 BM specimens.

Results: Although the overall RNA yield (normalized to 1 x 107 leukocytes) was not different, the RNA preparation using unstabilized reference samples had an ~3 times higher failure rate. With the PAXgene system, we observed significantly higher RNA integrity compared with the reference RNA preparation system (P <0.01). In the stabilized samples, we found very high pairwise correlation in gene expression ({Delta}{Delta}CT 0.16–0.53) for the analyzed genes (GATA1, RUNX1, NCAM1, and SPI1) after 48-h storage compared with immediate preparation of RNA (2 h after BM collection). However, we found major differences in half of the analyzed genes using the reference RNA isolation procedure ({Delta}{Delta}CT 1.07 and 1.32).

Conclusions: The PAXgene system is able to stabilize RNA from clinical BM samples and is suitable to isolate high-quality and -quantity RNA.


   Introduction
Top
 Abstract
 Introduction
Patients, Materials, and Methods
 Results
 Discussion
 References
 
Gene expression analysis by quantitative reverse-transcription PCR (Q-RT-PCR)1 is widely used in basic and clinical research of cancer. In patients with leukemia, bone marrow (BM) specimens—rather than peripheral blood (PB) specimens—are the source of material for initial diagnosis and follow-up evaluation of treatment response. Nearly all children with leukemia in Germany (96.9%;http://www.kinderkrebsregister.de ) are treated in nationwide multicenter clinical trials with centralized laboratories for diagnosis and research questions. Anticoagulated BM specimens are commonly shipped to these laboratories by overnight mail at room temperature. Owing to gene induction, repression, and RNA degradation, storage of native specimens of PB or BM and method of RNA isolation have significant influence on gene expression (1)(2)(3). However, the evaluation of gene expression levels using Q-RT-PCR or microarray technology is essential for the investigation of the molecular origin of leukemia (4)(5)(6)(7)(8) as well as for monitoring minimal residual disease (9).

Acute leukemias are clonal disorders of the hematopoietic system that arise in the BM. The current hypothesis for leukemogenesis published by Bonnet and Dick (10) assumes a multistep process starting from a leukemic stem cell that undergoes hierarchical differentiation into leukemic blasts. The aberrant expression of different transcription factors that are crucial at specific stages of hematopoiesis is believed to have a dominant role in either generation or maintenance of the malignant clone. The RUNX1 gene (runt-related transcription factor 1)2 is the most frequent target gene of aberrant activity in human leukemias. It is involved in several chromosomal translocations, e.g., t(8;21) in childhood acute myeloid leukemia, t(12;21) in childhood acute lymphoblastic leukemia, and t(3;21) in chronic myeloid leukemia or myelodysplastic syndrome. Furthermore, a high incidence of RUNX1 point mutations has been revealed. Recently, a 3rd mode of RUNX1 involvement in leukemogenesis—an increased dosage of RUNX1—has been reported (11). The adhesion molecule NCAM1 (neural cell adhesion molecule 1) is commonly expressed at the surface of subsets of acute leukemias and furthermore contributes to hematopoiesis-supporting capacity of stromal cell lines (12). GATA1 (GATA binding protein 1) is essential for normal erythropoiesis, with a crucial role in cell survival and maturation (13). Mutations in exon 2 of GATA1, which are translated into a shorter GATA1s protein, are detectable in virtually all cases of myeloid leukemia of Down syndrome and transient myeloproliferative disease of newborns with Down syndrome (14). PU.1, which is encoded by the gene SPI1 (spleen focus forming virus proviral integration oncogene 1), serves as a suppressor of acute myeloid leukemia, and its down-regulation results in an aggressive form of acute myeloid leukemia (15)(16)(17).

Recently, a commercially available in vitro diagnostic system for the stabilization of RNA in PB specimens (PAXgeneTM Blood RNA System, PreAnalytiX) arrived on the market. This system inhibits RNA degradation and prevents samples from ex vivo changes in gene expression starting immediately at the time of sample collection (18). RNA stabilization is a major prerequisite for reliable transcript analysis in whole blood samples. An important difference between PB and BM that is relevant for RNA stabilization is BM’s significantly higher range in cellularity, especially in diagnostic samples of acute leukemias (>500 000 leukocytes/µL), thus defining a strong need to adapt the existing PAXgene Blood RNA system to BM. For research use only, PreAnalytiX has optimized the system for BM. The system enables the collection, stabilization, storage, and transportation of human BM specimens, together with a rapid and efficient protocol for isolation and purification of intracellular RNA.

Our aim was to investigate the influence of preanalytical steps of sample handling after aspiration and before RNA analysis, focusing on RNA yield and integrity and stability of gene expression profiles. We compared the PAXgene Bone Marrow RNA System with a method without RNA stabilization using standard tubes and considered a 2-day storage of BM, reflecting the average shipping time in common clinical settings. We used transcript concentrations of marker genes GATA1, RUNX1, SPI1, and NCAM1, commonly involved in leukemogenesis, to study the need for stabilization of BM samples in a clinical setting of disease monitoring.


   Patients, Materials, and Methods
Top
 Abstract
 Introduction
 Patients, Materials, and Methods
Results
 Discussion
 References
 
patients and samples
We collected BM specimens (n = 64), using 5000 IU heparin per 5 mL BM as anticoagulant, from randomly selected children with acute leukemia at initial diagnosis or during treatment or from children without pathologic hematopoietic findings. BM specimens were obtained after informed consent from each patient or patient’s guardian. All investigations were approved by the local ethics committee. Immediately after collection, we transferred 2.5 mL BM to 2 PAXgene Bone Marrow RNA tubes containing optimized RNA stabilization solution. After 2 h (P2) or 48 h (P48) of storage at room temperature after collection, the tubes were frozen (–20 °C) for a median of 5 weeks (range, 1–15 weeks). After thawing, we prepared RNA by use of the PAXgene Bone Marrow RNA Kit. Two specimens served as reference samples: 0.5 mL BM was transferred into sterile plastic tubes and processed using the QIAamp® RNA Blood Mini Kit (Qiagen) at the same time points [2 h (R2) and 48 h (R48); Fig. 1 ]. The time points were chosen to represent the operational procedures in many hospitals and laboratories. For the reference protocol, we took into account a lag of 2 h (for in-house transportation and sample receipt in the laboratory) between collection of BM and preparation of RNA in the laboratory. For the PAXgene system, a minimum precipitation time of 2 h is required before the RNA precipitate can be centrifuged. The 48-h storage at room temperature of either the plastic tube with anticoagulated BM (reference protocol) or the PAXgene Bone Marrow RNA tube represents the time interval between collection of BM and shipping to an external laboratory.


Figure 1
View larger version (32K):
[in this window]
[in a new window]

  		Figure 1. Flowchart of the study design.

Immediately after collection, 2.5 mL anticoagulated BM was transferred to 2 PAXgene Bone Marrow RNA tubes containing optimized RNA stabilization solution. After 2 (P2) and 48 (P48) h of storage at room temperature after collection, the tubes were frozen (–20 °C) for a median of 5 weeks (range, 1–15 weeks). After thawing, RNA was prepared using the PAXgene Bone Marrow RNA Kit. Two other specimens of 0.5 mL BM each were transferred into sterile plastic tubes as reference samples and were processed using the QIAamp RNA Blood Mini Kit (Qiagen) at the same time points (R2 and R48).

BM specimens were eligible for evaluation if all 4 RNA preparation procedures (2 PAXgene samples and 2 reference samples) fulfilled the following selection criteria for downstream analysis: (a) RNA concentration >5 mg/L and (b) RNA integrity number (RIN) >7 or A260/A280 between 1.9 and 2.1. We measured leukocytes in BM specimens by use of FlowCountTM Fluorospheres (Beckman Coulter) and a Cytomics FC 500 flow cytometer (Beckman Coulter).

rna preparation procedure
We isolated and purified RNA from PAXgene Bone Marrow RNA tubes using the PAXgene Bone Marrow RNA Kit according to manufacturer’s instructions. In brief, we harvested nucleic acids by centrifugation of collection tubes. We washed, resuspended, and incubated pellets in optimized buffers together with proteinase K. Cell lysates were homogenized and cell debris removed by use of PAXgene Shredder columns. RNA binding conditions were adjusted with ethanol, and lysates were applied to RNA spin columns. After centrifugation through a silica-gel membrane, we removed contaminants with efficient wash steps, followed by DNase I treatment to remove traces of bound DNA. After additional wash steps, RNA was released in 80 µL elution buffer and heat-denatured. We prepared reference RNA samples from unstabilized BM aliquots from sterile standard plastic tubes using the QIAamp RNA Blood Mini Kit as described in the handbook, with an elution volume of 50 µL.

rna yield and integrity
We diluted aliquots of RNA samples in 10 mmol/L Tris · Cl, pH 7.5, and quantified them using UV spectroscopy. We also used the buffers in which the RNA were isolated to zero the spectrophotometer in the final dilution of RNA aliquots to be quantified. We analyzed RNA integrity from aliquots of 1.5 µL RNA by use of RNA 6000 Nano reagents and chips on a Bioanalyzer 2100 device equipped with BioSizing software version A02.12 (Agilent Technologies). We applied the software RIN calculation algorithm to RNA fluorescence profiles after separation of RNA by capillary gel electrophoresis to establish RNA integrity with a score of 0 to 10 points (low to high RNA integrity).

q-rt-pcr
Q-RT-PCR was performed in a 1-step method with 1 ng total RNA by use of the Quantitect® SYBR® Green reverse-transcription (RT)-PCR Kit and Quantitect Primer Assays (Qiagen) for the detection of GATA1, RUNX1, NCAM1, and SPI1 and for control gene 18S rRNA. We chose 18S rRNA because general RNA degradation correlates very well with decreased amounts of 18S rRNA. We performed all assays in duplicate by use of the ABITM 7300 Real-Time PCR System (Applied Biosystems). Calculation was done by relative quantification using the 2–{Delta}{Delta}CT T method (19). Only those data with a CT value <35 (cutoff value) were included in the evaluation.

feasibility study
Before the above study, we performed a feasibility study with a subset of 31 BM specimens. Along with the quantification of RNA yield and RNA integrity, we analyzed the expression of IL8 gene (interleukin 8) using the Quantitect SYBR Green RT-PCR Kit (Qiagen) on the LightCycler® 2.0 System (Roche Applied Science). We chose IL8 because of its known up-regulation after short-term storage of clinical samples (20).


   Results
Top
 Abstract
 Introduction
 Patients, Materials, and Methods
 Results
Discussion
 References
 
Independent of the leukocyte count of the BM specimens (median, 17 151 leukocytes/µL; range, 2800–546 740), the handling of the PAXgene Bone Marrow RNA System was easy and convenient. There was no association between leukocyte count and the yield of total RNA in either preparation method (data not shown). Regarding the influence of 2-day storage on IL8 gene expression, we found a huge difference in the unstabilized samples compared with the stabilized samples [median change in gene expression, 14.8-fold (R) vs 0.7-fold (P)]. As shown in Fig. 2 , the CT values of the R48 samples were consistently lower than the CT values of the R2 samples, representing an increase in gene expression. Although the correlation between CT values of P2 and P48 is not optimal (slope, 0.90; R2 = 0.64), possibly because of missing normalization with transcripts of a housekeeping gene, the correlation for the reference samples was even worse (slope, 0.32; R2 = 0.08).


Figure 2
View larger version (27K):
[in this window]
[in a new window]

  		Figure 2. IL8 gene expression in RNA samples prepared from BM with 48 and 2 h of storage using either the PAXgene system (P) or the reference protocol without RNA stabilization (R).

The diagrams show the cycle threshold (CT) of the Q-RT-PCR of IL8 for the samples at 2 (x axis) and 48 (y axis) h of storage for either preparation method. The black diagonal line represents an ideal correlation, i.e., no change in gene expression between the 2 time points; the dashed line indicates the calculated regression line (P, y = 0.90 · x, R2 = 0.64; R, y = 0.31 · x, R2 = 0.08).There is a high increase in IL8 expression after 48-h storage for the RNA prepared by the reference method compared with the PAXgene method.

yield and integrity of rna
Altogether, 180 RNA samples, processed from 45 BM specimens, fulfilled the inclusion criteria and were analyzed for yield, integrity, and gene expression profiles. We excluded 19 specimens because of low-yield or low-quality RNA samples: P2 (n = 3), P48 (n = 5), R2 (n = 12), and R48 (n = 15). In 26 samples, criteria for both yield and quality were not fulfilled, 6 samples were of bad quality, and 3 samples (all reference samples) were of insufficient yield (1.2, 1.2, and 2.6 mg/L, respectively).

The number of invalid samples obtained with the reference method was ~3 times higher than with the PAXgene system (27 vs 8 samples), demonstrating higher overall RNA quality and quantity with PAXgene.

The RNA yield for each method was measured and normalized to 1 x 107 leukocytes in the input BM sample. As shown in Fig. 3A , the overall yield was similar in the 4 RNA isolation procedures. We found no differences in RNA yield at 2 and 48 h for each method separately (ANOVA).


Figure 3
View larger version (60K):
[in this window]
[in a new window]

  		Figure 3. RNA yield and integrity.

(A), RNA yield of BM aliquots with 2 and 48 h of storage at room temperature obtained using the PAXgene system (P) and the reference protocol without RNA stabilization (R) (mean +SE). (B), box plot of RIN values from RNA samples of BM aliquots with 2 and 48 h of storage at room temperature obtained using the PAXgene system (P) and the reference protocol without RNA stabilization (R) (black bars, median; gray rectangles, 25th to 75th percentile; black lines, 5th to 95th percentile; rhombs, minimum/maximum value). (C), representative RNA integrity analysis of 4 RNA samples of a single BM aliquot with 2 and 48 h of storage at room temperature obtained using the PAXgene system (P) and the reference protocol without RNA stabilization (R).

The integrity of the isolated RNA using the PAXgene system was significantly higher at both time points compared with the reference system: P2, 8.6 (0.2) vs R2, 6.8 (0.4), P = 0.0003, and P48, 8.1 (0.2) vs R48 6.7 (0.5), P = 0.008. We found no statistically significant difference between R2 and R48, but a slight decrease between P2 and P48 (P = 0.048). As shown in Fig. 3BUp , the spreading of RIN is more distinct in the RNA prepared by the reference method with regard to the 25% to 75% confidence interval: R2, 5.2–9.1 and R48, 4.1–9.0 vs P2 8.4–9.3 and P48, 7.8–8.9. As an example, Fig. 3CUp illustrates the RNA integrity of a complete data set with 4 samples (P2, P48, R2, and R48).

paxgene stabilizes gene expression profiles
We analyzed mRNA levels of different genes after RNA preparation using the PAXgene procedure at P2 and P48. Differences of {Delta}{Delta}CT, calculated as {Delta}CT(P2) – {Delta}CT(P48) for the control gene (18S rRNA), were homogeneous within a tight range: GATA1 0.17 (0.10), RUNX1 0.27 (0.17), NCAM1 0.53 (0.23), and SPI1 0.16 (0.09). This results in a high pairwise correlation in gene expression for any gene at P48 compared with P2: GATA1 89 (6)%, RUNX1 83 (10)%, NCAM1 69 (12)%, and SPI1 89 (6)% (Fig. 4 ).


Figure 4
View larger version (15K):
[in this window]
[in a new window]

  		Figure 4. Change of gene expression in RNA samples prepared from BM with 48 and 2 h of storage using either the PAXgene system (P) or the reference protocol without RNA stabilization (R).

Data are shown as mean (SE).

gene-dependent change of expression level using standard rna isolation techniques
Regarding expression levels of the same genes after RNA preparation from unstabilized BM samples using QIAamp RNA Blood Kit, the results are more heterogeneous. For 2 of the 4 analyzed genes, the differences in {Delta}{Delta}CT, {Delta}CT(R2) – {Delta}CT(R48), were also within the range of ±0.5, whereas for the 2 other genes, the differences were >1: GATA1 1.32 (0.19), RUNX1 0.25 (0.18), NCAM1 1.07 (0.23), and SPI1 –0.01 (0.14). The resulting expression levels at R48 for the latter genes were accordingly reduced to 40 (6)%, P <0.0001 (GATA1) and 47 (8)%, P = 0.005 (NCAM1; Fig. 4Up ).

expression levels are not comparable using different rna isolation protocols
When we compared the 2 RNA isolation procedures with each other, there were major differences in the expression levels of the analyzed genes. We observed lower mean (SD) gene expression at P2 vs R2 for all genes: GATA1 70 (12)%, RUNX1 50 (11)%, NCAM1 41 (8)%, and SPI1 51 (6)%. At the 48-h time point, the changes in gene expression for RUNX1 and SPI1 were similar at 45 (7)% and 43 (6)%, respectively, whereas for the other 2 genes (GATA1 and NCAM1), the decrease in gene expression using the reference method resulted in significant discrepancy: 138 (26)% and 76 (16)% (Fig. 5 ).


Figure 5
View larger version (17K):
[in this window]
[in a new window]

  		Figure 5. Differences of transcript levels between RNA samples prepared from BM with the PAXgene system and the reference protocol without RNA stabilization at same test time point with 2 and 48 h of storage.

Data are shown as mean (SE).


   Discussion
Top
 Abstract
 Introduction
 Patients, Materials, and Methods
 Results
 Discussion
References
 
In multicenter clinical trials, a centralized analysis of patient samples is generally performed to minimize interlaboratory variations by applying a standardized method to all samples. Little is known, however, about the impact of preanalytical sample handling, particularly for molecular genetic analyses based on mRNA. High interindividual and temporal variations of gene expression patterns have been demonstrated because of genetic background, age, sex, and the time of day at which the sample was taken (21). Other similarly important variables affecting gene expression profiles include mode of sample aspiration, use of different anticoagulants, variable temperature during storage, and time frame between aspiration and processing of the sample.

We carried out a comparative study of a newly developed system for RNA stabilization in whole BM specimens (PAXgene Bone Marrow RNA System) and unstabilized controls after different times of storage. The time delays before RNA preparation were chosen to represent the average shipping time between clinics and laboratories. Although alterations in gene expression may occur during a very short period of time, our study was designed to represent realistic operational procedures in a hospital, where BM punctures (especially in children) are often performed in an operating room, and a routine in-house transport time to the laboratory has to be taken into account.

The promising data from the feasibility study confirmed the known up-regulation of IL8 gene expression in unstabilized specimens. The PAXgene-stabilized BM specimens with 48 and 2 h of storage showed high accordance in terms of IL8 gene expression, prompting us to perform a more comprehensive study. The analyses of purity (calculated as A260/A280 ratio) and RNA integrity (calculated by RIN) served as tools to determine the overall quality of the RNA preparation and purification procedure in terms of purity and possible degree of overall RNA degradation. Although the use of cDNA microarrays allows genomewide gene expression screening, their value for exact quantification is limited (22). For more detailed information about the stabilization of RNA, Q-RT-PCR was performed to quantify mRNA concentrations. The genes to be analyzed were selected because of their attributes as key regulators during different stages of hematopoiesis and leukemogenesis, as well as their more or less distinct organ specificity: NCAM1 and RUNX1 as representatives of genes with functions (hematopoiesis, osteogenesis, cell adhesion) on many cell types (hematopoietic cells, neurons, muscles) of various differentiated tissues (embryonic cells, differentiated cells), GATA1 and SPI1 having more circumscribed compartments and cell types (erythropoiesis/megakaryopoiesis, myelopoiesis, and lymphopoiesis).

There were no major differences in the yield of isolated RNA between the 4 different procedures. Regarding the integrity of the RNA, however, the PAXgene system was superior to the reference method of unstabilized BM samples at both time points. Handling of samples with very high leukocyte counts was not impaired compared with samples with normal leukocyte counts. The unexpectedly high integrity of the RNA samples obtained after 48 h of storage under unstabilized conditions (R48) might be explained by the RNA preparation procedure: in contrast to a direct preparation from whole BM, the QIAamp procedure used here includes erythrocyte lysis as well as the generation of a cell pellet that contains only intact white BM cells. Therefore, possible RNA degradation products are removed with the supernatant.

Regarding gene expression profiling, the use of RNA stabilization reagents is still controversial: there are some reports of differences in gene expression resulting from poor stability of genes analyzed using either microarray technology (23) or Q-RT-PCR (24)(25). In terms of RNA quality and gene expression, however, our results from BM samples show excellent pairwise correlation between different incubation times using quantitative RT-PCR and are in line with those derived from PB (3)(26)(27). The PAXgene system is appropriate for quantification of gene expression levels in BM samples. For some genes, however, the reference method resulted in similar gene expression levels, so the use of a stabilization reagent does not seem to be essential for every gene. This has also been shown for the quantification of tissue factor (F3) and vascular endothelial growth factor, with comparable results derived from stabilized and unstabilized samples (28). Therefore, each researcher must carefully evaluate the preanalytical handling of the samples and choose a system for RNA ex vivo stabilization in case of gene expression analysis in later project phases with unknown targets at start of study. In studies where RNA isolates are stored for a long time before analysis of currently unrevealed disease marker transcripts, BM stabilization using the PAXgene system is recommended.

Different principles of RNA isolation may lead to different gene expression levels: whereas the QIAamp RNA isolation procedure includes erythrocyte lysis, during the PAXgene protocol, the RNA of all BM cells is isolated. Therefore, it is not suitable to directly compare mRNA expression levels derived from different isolation procedures.

In conclusion, the PAXgene Bone Marrow RNA System is suitable to stabilize, isolate, and purify RNA in high quality and quantity from clinical BM samples. The ability to preserve gene expression is comparable to that of PB specimens reported in detail by different researchers with the PAXgene Blood RNA System.


   Acknowledgments
 
We thank Carolin Augsburg and Heike Balven for excellent technical assistance and Douglas McGarvey for review of the manuscript. Conflicts of interest: K.G. and J.L. are full-time employees and participate in the stock options program of Qiagen GmbH.


   Footnotes
 
1 Nonstandard abbreviations: Q-RT, quantitative reverse-transcription; RT, reverse-transcription; BM, bone marrow; PB, peripheral blood; RIN, RNA integrity number.

2 Human genes: RUNX1, runt-related transcription factor 1; GATA1, GATA binding protein 1; SPI1, spleen focus forming virus proviral integration oncogene 1; NCAM1, neural cell adhesion molecule 1.


   References
Top
 Abstract
 Introduction
 Patients, Materials, and Methods
 Results
 Discussion
 References
 

  1. Breit S, Nees M, Schaefer U, Pfoersich M, Hagemeier C, Muckenthaler M, et al. Impact of pre-analytical handling on bone marrow mRNA gene expression. Br J Haematol 2004;126:231-243.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  2. Feezor RJ, Baker HV, Mindrinos M, Hayden D, Tannahill CL, Brownstein BH, et al. Whole blood and leukocyte RNA isolation for gene expression analyses. Physiol Genomics 2004;19:247-254.[Abstract/Free Full Text]
  3. Chai V, Vassilakos A, Lee Y, Wright JA, Young AH. Optimization of the PAXgene blood RNA extraction system for gene expression analysis of clinical samples. J Clin Lab Anal 2005;19:182-188.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  4. Armstrong SA, Staunton JE, Silverman LB, Pieters R, den Boer ML, Minden MD, et al. MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia. Nat Genet 2002;30:41-47.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  5. Bourquin JP, Subramanian A, Langebrake C, Reinhardt D, Bernard O, Ballerini P, et al. Identification of distinct molecular phenotypes in acute megakaryoblastic leukemia by gene expression profiling. Proc Natl Acad Sci U S A 2006;103:3339-3344.[Abstract/Free Full Text]
  6. Golub TR, Slonim DK, Tamayo P, Huard C, Gaasenbeek M, Mesirov JP, et al. Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science 1999;286:531-537.[Abstract/Free Full Text]
  7. Moos PJ, Raetz EA, Carlson MA, Szabo A, Smith FE, Willman C, et al. Identification of gene expression profiles that segregate patients with childhood leukemia. Clin Cancer Res 2002;8:3118-3130.[Abstract/Free Full Text]
  8. Yeoh EJ, Ross ME, Shurtleff SA, Williams WK, Patel D, Mahfouz R, et al. Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling. Cancer Cell 2002;1:133-143.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  9. Viehmann S, Teigler-Schlegel A, Bruch J, Langebrake C, Reinhardt D, Harbott J. Monitoring of minimal residual disease (MRD) by real-time quantitative reverse transcription PCR (RQ-RT-PCR) in childhood acute myeloid leukemia with AML1/ETO rearrangement. Leukemia 2003;17:1130-1136.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  10. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997;3:730-737.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  11. Osato M, Ito Y. Increased dosage of the RUNX1/AML1 gene: a third mode of RUNX leukemia?. Crit Rev Eukaryot Gene Expr 2005;15:217-228.[ISI][Medline] [Order article via Infotrieve]
  12. Wang X, Hisha H, Taketani S, Inaba M, Li Q, Cui W, et al. Neural cell adhesion molecule contributes to hemopoiesis-supporting capacity of stromal cell lines. Stem Cells 2005;23:1389-1399.[Abstract/Free Full Text]
  13. Weiss MJ, Orkin SH. GATA transcription factors: key regulators of hematopoiesis. Exp Hematol 1995;23:99-107.[ISI][Medline] [Order article via Infotrieve]
  14. Wechsler J, Greene M, McDevitt MA, Anastasi J, Karp JE, Le Beau MM, et al. Acquired mutations in GATA1 in the megakaryoblastic leukemia of Down syndrome. Nat Genet 2002;32:148-152.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  15. Cook WD, McCaw BJ, Herring CD, John DL, Foote SJ, Nutt SL, et al. PU. 1 is a suppressor of myeloid leukemia, inactivated in mice by gene deletion and mutation of its DNA-binding domain. Blood 2004;104:3437-3444.[Abstract/Free Full Text]
  16. Stirewalt DL. Fine-tuning PU. 1. Nat Genet 2004;36:550-551.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  17. Rosenbauer F, Wagner K, Kutok JL, Iwasaki H, Le Beau MM, Okuno Y, et al. Acute myeloid leukemia induced by graded reduction of a lineage-specific transcription factor, PU. 1. Nat Genet 2004;36:624-630.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  18. Rainen L, Oelmueller U, Jurgensen S, Wyrich R, Ballas C, Schram J, et al. Stabilization of mRNA expression in whole blood samples. Clin Chem 2002;48:1883-1890.[Abstract/Free Full Text]
  19. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25:402-408.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  20. Tanner MA, Berk LS, Felten DL, Blidy AD, Bit SL, Ruff DW. Substantial changes in gene expression level due to the storage temperature and storage duration of human whole blood. Clin Lab Haematol 2002;24:337-341.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  21. Whitney AR, Diehn M, Popper SJ, Alizadeh AA, Boldrick JC, Relman DA, et al. Individuality and variation in gene expression patterns in human blood. Proc Natl Acad Sci U S A 2003;100:1896-1901.[Abstract/Free Full Text]
  22. Wang Y, Barbacioru C, Hyland F, Xiao W, Hunkapiller KL, Blake J, et al. Large scale real-time PCR validation on gene expression measurements from two commercial long-oligonucleotide microarrays. BMC Genomics 2006;7:59.[CrossRef][Medline] [Order article via Infotrieve]
  23. Thach DC, Lin B, Walter E, Kruzelock R, Rowley RK, Tibbetts C, et al. Assessment of two methods for handling blood in collection tubes with RNA stabilizing agent for surveillance of gene expression profiles with high density microarrays. J Immunol Methods 2003;283:269-279.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  24. Thorn I, Olsson-Stromberg U, Ohlsen C, Jonsson AM, Klangby U, Simonsson B, et al. The impact of RNA stabilization on minimal residual disease assessment in chronic myeloid leukemia. Haematologica 2005;90:1471-1476.[ISI][Medline] [Order article via Infotrieve]
  25. Kagedal B, Lindqvist M, Farneback M, Lenner L, Peterson C. Failure of the PAXgene Blood RNA System to maintain mRNA stability in whole blood. Clin Chem Lab Med 2005;43:1190-1192.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  26. Wang J, Robinson JF, Khan HM, Carter DE, McKinney J, Miskie BA, et al. Optimizing RNA extraction yield from whole blood for microarray gene expression analysis. Clin Biochem 2004;37:741-744.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  27. Spijker S, van de Leemput JC, Hoekstra C, Boomsma DI, Smit AB. Profiling gene expression in whole blood samples following an in-vitro challenge. Twin Res 2004;7:564-570.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  28. Isaksson HS, Nilsson TK. Preanalytical aspects of quantitative TaqMan real-time RT-PCR: Applications for TF and VEGF mRNA quantification. Clin Biochem 2006;39:373-377.[CrossRef][Medline] [Order article via Infotrieve]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
clinchem.2006.078592v1
53/4/587    most recent
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Langebrake, C.
Right arrow Articles by Reinhardt, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Langebrake, C.
Right arrow Articles by Reinhardt, D.
Related Collections
Right arrow Molecular Diagnostics and Genetics

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS