Background: Accurate quantification of mRNA in whole blood is made difficult by the simultaneous degradation of gene transcripts and unintended gene induction caused by sample handling or uncontrolled activation of coagulation. This study was designed to compare a new blood collection tube (PAXgeneTM Blood RNA System) and a companion sample preparation reagent set with a traditional sample collection and preparation method for the purpose of gene expression analysis. Methods: We collected parallel blood samples from healthy donors into the new sample collection tubes and control EDTA tubes and performed serial RNA extractions on samples stored for 5 days at room temperature and for up to 90 days at 4 and 20 °C. Samples were analyzed by Northern blot analysis or reverse transcription-PCR (RT-PCR). Results: Specific mRNA concentrations in blood stored in EDTA tubes at any temperature changed substantially, as determined by high-precision RT-PCR. These changes were eliminated or markedly reduced when whole blood was stored in PAXgene tubes. Loss of specific mRNAs, as measured by RT-PCR, reflected total RNA depletion as well as specific mRNA destruction demonstrated by Northern blot analysis. The salutary effects of PAXgene on mRNA stabilization extended to blood samples from eight unrelated donors. Conclusions: Compared with whole blood collected in EDTA tubes and extracted by an organic method, the PAXgene Blood RNA System reduced RNA degradation and inhibited or eliminated gene induction in phlebotomy whole blood samples. Storage of whole blood samples in PAXgene tubes can be recommended for clinically related blood samples that will be analyzed for total or specific RNA content.
Preanalytical handling of tissue samples can influence bioanalyte quality and ultimately outcome of analytical results. The aim of this study was to compare RNA quality, performance in real time RT PCR and histology of formalin-fixed tissue to that of tissue fixed and stabilized with a formalin-free fixative, the PAXgene Tissue System (PAXgene), in an animal model under highly controlled preanalytical conditions. Samples of rat liver, kidney, spleen, intestine, lung, heart muscle, brain, and stomach tissue were either fixed in formalin or fixed in PAXgene or fresh frozen in liquid nitrogen. RNA was extracted from all samples, examined for integrity in microcapillary electrophoresis, and used in a series of quantitative RT PCR assays with increasing amplicon length. Histology of paraffin-embedded samples was determined by staining with hematoxylin and eosin. Histology of all formalin-fixed and PAXgene fixed samples was comparable. RNA with acceptable integrity scores could be isolated from all embedded tissues, 4.0 to 7.2 for formalin and 6.4 to 7.7 for PAXgene, as compared to 8.0 to 9.2 for fresh frozen samples. While RNA with acceptable RINs (RNA integrity number) could be isolated from formalin-fixed samples, in microcapillary electrophoresis this RNA separated with a slower migration rate and displayed diffuse, less focused peaks for ribosomal RNA as compared to RNA from frozen or PAXgene fixed samples. Furthermore, RNA from formalin-fixed tissues exhibited inhibition in quantitative RT PCR assays which increased with increasing amplicon length, while RNA from PAXgene fixed samples did not show such inhibition. In conclusion, our results demonstrate that excluding other preanalytical factors, PAXgene Tissue System preserves histology similarly to formalin, but unlike formalin, does not chemically modify RNA. RNA purified from PAXgene fixed tissues is of high integrity and performs as well as RNA from fresh frozen tissue in RT PCR regardless of amplicon length.
For accurate diagnosis, prediction of outcome, and selection of appropriate therapies, the molecular characterization of human diseases requires analysis of a broad spectrum of altered biomolecules, in addition to morphological features, in affected tissues such as tumors. In a high-throughput screening approach, we have developed the PAXgene Tissue System as a novel tissue stabilization technology. Comprehensive characterization of this technology in stabilized and paraffin-embedded human tissues and comparison with snap-frozen tissues revealed excellent preservation of morphology and antigenicity, as well as outstanding integrity of nucleic acids (genomic DNA, miRNA, and mRNA) and phosphoproteins. Importantly, PAXgene-fixed, paraffin-embedded tissues provided RNA quantity and quality not only significantly better than that obtained with neutral buffered formalin, but also similar to that from snap-frozen tissue, which currently represents the gold standard for molecular analyses. The PAXgene tissue stabilization system thus opens new opportunities in a variety of molecular diagnostic and research applications in which the collection of snap-frozen tissue is not feasible for medical, logistic, or ethical reasons. Furthermore, this technology allows performing histopathological analyses together with molecular studies in a single sample, which markedly facilitates direct correlation of morphological disease phenotypes with alterations of nucleic acids and other biomolecules.
The diagnostic use of in vitro molecular assays can be limited by the lack of guidelines for collection, handling, stabilization and storage of patient specimens. One of the major goals of the EC funded project SPIDIA (www.spidia.eu) is to develop evidence-based quality guidelines for the pre-analytical phase of blood samples used for molecular testing which requires intracellular RNA analytes. To this end, a survey and a pan-European external quality assessment (EQA) were implemented. This report is the summary of the results of that trial. With the European Federation of Laboratory Medicine (EFLM) support, 124 applications for participation in the trial were received from 27 different European countries, and 102 laboratories actually participated in the trial. Each participating laboratory described their respective laboratory policies and practices as well as blood collection tubes typically used in performing this type of testing. The participating laboratories received two identical blood specimens: in an EDTA tubes (unstabilized blood; n=67) or in tubes designed specifically for the stabilization of intracellular RNA in blood (PAXgene® Blood RNA tubes; n=35). Laboratories were requested to perform RNA extraction according to the laboratory's own procedure as soon as possible upon receipt of the tubes for one tube and 24h after the first extraction for the second tube. Participants (n=93) returned the two extracted RNAs to SPIDIA facility for analysis, and provided details about the reagents and protocols they used for the extraction. At the SPIDIA facility responsible for coordinating the study, the survey data were classified, and the extracted RNA samples were evaluated for purity, yield, integrity, stability, and the presence of interfering substances affecting RT-qPCR assays. All participants received a report comparing the performance of the RNA they submitted to that of the other participants. All the results obtained by participants for each RNA quality parameter were classified as "in control", "warning", "out of control" and "missing" by consensus mean analysis. From the survey data, the most variable parameters were the volume of blood collected and the time and storage temperature between blood collection and RNA extraction. Analyzing the results of quality testing of submitted RNA samples we observed a data distribution of purity, yield, and presence of assay interference in agreement with expected values. The RNA Integrity Number (RIN) values distribution was, on the other hand, much wider than the optimal expected value, which led to an "in control" classification, even for partly degraded RNA samples. On the other hand, RIN values below 5 significantly correlated with a reduction of GAPDH expression levels. Furthermore, the distribution of the values of the four transcripts investigated (c-fos, IL-1β, IL-8, and GAPDH) was wide and the RNA instability between samples separated by 24h were similar. Assuming the presence of at least two quality parameters "out of control" as an indication of a c...
Exploiting the differential expression of genes for Calvin cycle enzymes in bundle-sheath and mesophyll cells of the C4 plant Sorghum bicolor L., we isolated via subtractive hybridization a molecular probe for the Calvin cycle enzyme D-ribulose-5-phosphate 3-epimerase (R5P3E)(EC 5.1.3.1), with the help of which several full-size cDNAs were isolated from spinach. Functional identity of the encoded mature subunit was shown by R5P3E activity found in affinity-purified glutatione S-transferase fusions expressed in Escherichia coli and by three-fold increase of R5P3E activity upon induction of E. coli overexpressing the spinach subunit under the control of the bacteriophage T7 promoter, demonstrating that we have cloned the first functional ribulose-5-phosphate 3-epimerase from any eukaryotic source. The chloroplast enzyme from spinach shares about 50% amino acid identity with its homologues from the Calvin cycle operons of the autotrophic purple bacteria Alcaligenes eutrophus and Rhodospirillum rubrum. A R5P3E-related eubacterial gene family was identified which arose through ancient duplications in prokaryotic chromosomes, three R5P3E-related genes of yet unknown function have persisted to the present within the E. coli genome. A gene phylogeny reveals that spinach R5P3E is more similar to eubacterial homologues than to the yeast sequence, suggesting a eubacterial origin for this plant nuclear gene.
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