Biomedical subjects

P Lambin

Publications and source records attributed to P Lambin.

At least 19 recordsLinked to original sources

PubMed2006

[Protons and ions in the treatment of cancer; a systematic review of the literature].

OBJECTIVE: To provide an overview of the present role of proton and ion therapy, also referred to as 'charged particle therapy', in the treatment of cancer. DESIGN: Systematic literature study. METHOD: Systematic electronic searches were carried out in 12 databases according to the Cochrane Collaboration criteria, without restriction as to year of publication or study design. Manual searches of bibliographies and journals were also performed. The inclusion criteria were: at least 20 patients and a follow-up of at least 2 years. In addition, experts on the subject were consulted by correspondence for their opinion. RESULTS: The search identified 36 relevant articles on proton therapy and 15 on ion therapy. Based on prospective and retrospective studies, proton irradiation emerged as the treatment of choice for ocular tumours, chordomas and skull-base tumours. For prostate cancer, the results were comparable with the best results of photon therapy. Ion therapy was still in an experimental phase. CONCLUSION: According to the current literature, proton therapy is looked upon as the preferred treatment modality for certain rare tumours, such as ocular tumours, chordoma, and skull-base tumours. However, charged particle therapy as a whole, and especially ion therapy, is not supported as the treatment of choice for cancer by published evidence. Nevertheless, the potential theoretical benefit of this treatment is great.

Chordoma↗

PubMed2006

Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo.

The unique properties of the tumour microenvironment can be exploited by using recombinant anaerobic clostridial spores as highly selective gene delivery vectors. Although several recombinant Clostridium species have been generated during the past decade, their efficacy has been limited. Our goal was to substantially improve the prospects of clostridia as a gene delivery vector. Therefore, we have assessed a series of nitroreductase (NTR) enzymes for their capacity to convert the innocuous CB1954 prodrug to its toxic derivative. Among the enzymes tested, one showed superior prodrug turnover characteristics. In addition, we established an efficient gene transfer procedure, based on conjugation, which allows for the first time genetic engineering of Clostridium strains with superior tumour colonisation properties with high success rates. This conjugation procedure was subsequently used to create a recombinant C. sporogenes overexpressing the isolated NTR enzyme. Finally, analogous to a clinical setting situation, we have tested the effect of multiple consecutive treatment cycles, with antibiotic bacterial clearance between cycles. Importantly, this regimen demonstrated that intravenously administered spores of NTR-recombinant C. sporogenes produced significant antitumour efficacy when combined with prodrug administration.

Animals↗

PubMed2006

Phased attenuation correction in respiration correlated computed tomography/positron emitted tomography.

The motion of lung tumors with respiration causes difficulties in the imaging with computed tomography (CT) and positronemitted tomography (PET). Since an accurate knowledge of the position of the tumor and the surrounding tissues is needed for radiation treatment planning, it is important to improve CT/PET image acquisition. The purpose of this study was to evaluate the potential to improve image acquisition using phased attenuation correction in respiration correlated CT/PET, where data of both modalities were binned retrospectively. Respiration correlated scans were made on a Siemens Biograph Sensation 16 CT/PET scanner which was modified to make a low pitch CT scan and list mode PET scan possible. A lollipop phantom was used in the experiments. The sphere with a diameter of 3.1 cm was filled with approximately 20 MBq 18F-FDG. Three longitudinal movement amplitudes were tested: 2.5, 3.9, and 4.8 cm. After collection of the raw CT data, list mode PET data, and the respiratory signal CT/PET images were binned to ten phases with the help of in-house-built software. Each PET phase was corrected for attenuation with CT data of the corresponding phase. For comparison, the attenuation correction was also performed with nonrespiration correlated (non-RC) CT data. The volume and the amplitude of the movement were calculated for every phaseof both the CT and PET data (with phased attenuation correction). Maximum and average activity concentrations were compared between the phased and nonphased attenuation corrected PET. With a standard non-RC CT/PET scan, the volume was underestimated by as much as 46% in CT and the PET volume was overestimated to 370%. The volumes found with RC-CT/PET scanning had average deviations of 1.9% (+/- 4.8%) and 1.5% (+/- 3.4%) from the actual volume, for the CT and PET volumes, respectively. Evaluation of the maximum activity concentration showed a clear displacement in the images with non-RC attenuation correction, and activity values were on average14% (+/- 12%) lower than with phased attenuation correction. The standard deviation of the maximum activity values found in the different phases was a factor of 10 smaller when phased attenuation correction was applied. In this phantom study, we have shown that a combination of respiration correlated CT/PET scanning with application of phased attenuation correction can improve the imaging of moving objects and can lead to improved volume estimation and a more precise localization and quantification of the activity.

Humans↗

PubMed2005

Systematic review and meta-analysis of randomised, controlled trials of the timing of chest radiotherapy in patients with limited-stage, small-cell lung cancer.

BACKGROUND: We undertook a systematic review and literature-based meta-analysis to determine whether the timing of chest radiotherapy may influence the survival of patients with limited-stage small-cell lung cancer (LS-SCLC). MATERIALS: Eligible randomised controlled clinical trials were identified according to the Cochrane Collaboration Guidelines, comparing different timing of chest radiotherapy in patients with LS-SCLC. Early chest irradiation was defined as beginning within 30 days after the start of chemotherapy. RESULTS: Considering all seven eligible trials, the overall survival at 2 or 5 years was not significantly different between early or late chest radiotherapy. When only trials were considered that used platinum chemotherapy concurrent with chest radiotherapy, a significantly higher 5-year survival was observed when chest radiotherapy was started within 30 days after the start of chemotherapy (2-year survival: OR: 0.73, 95% CI 0.51-1.03, P = 0.07; 5-year survival: OR: 0.64, 95% CI 0.44-0.92, P = 0.02). This was even more pronounced when the overall treatment time of chest radiotherapy was less than 30 days. CONCLUSIONS: There are indications that the 5-year survival rates of patients with LS-SCLC are in favour of early chest radiotherapy, with a significant difference if the overall treatment time of chest radiation is less than 30 days.

Carcinoma, Small Cell↗

PubMed2005

Early versus late chest radiotherapy for limited stage small cell lung cancer.

BACKGROUND: It is standard clinical practice to combine chemotherapy and chest radiotherapy in treating patients with limited-stage small cell lung cancer. However, the best way to integrate both modalities is unclear. OBJECTIVES: To establish the most effective way of combining chest radiotherapy with chemotherapy for patients with limited-stage small cell lung cancer in order to improve long-term survival. SEARCH STRATEGY: The electronic databases MEDLINE, EMBASE, Cancerlit and the Cochrane Central Register of Controlled Trials (CENTRAL), reference lists, handsearching of journals and conference proceedings, and discussion with experts were used to identify potentially eligible trials, published and unpublished. SELECTION CRITERIA: Randomised controlled clinical trials comparing different timing of chest radiotherapy in patients with limited-stage small cell lung cancer. DATA COLLECTION AND ANALYSIS: Seven randomised trials were reviewed. There were differences in the timing and the overall treatment time of chest radiotherapy, the overall treatment time of , and the type of chemotherapy used. MAIN RESULTS: No significant differences in the 2-year and the 5-year survival were found, whether chest radiotherapy was delivered within 30 days after the start of chemotherapy or later. When the only study that delivered chest radiotherapy during cycles of non-platinum chemotherapy was excluded, a trend for the 5-year survival was observed (RR:0.93, p=0.07) in favour of early radiation, but not for the 2-year survival. Survival at 5 years, but not at 2 years, was significantly better for those having early chest radiotherapy delivered in an overall treatment time of less than 30 days compared with a longer treatment time (RR: 0.90, p=0.006). These results, however, should be interpreted with caution because the largest trial has follow-up data at three years, but not later. It remains to be seen what the effect of longer follow up will be for 5-year survival rates. Local tumour control was not significantly different between early and late chest radiotherapy. The incidence of severe pneumonitis or severe oesophagitis was not significantly different for early versus late thoracic radiotherapy. However, a trend for a higher chance to develop pneumonitis when early chest radiotherapy was delivered during non-platinum based chemotherapy was observed. AUTHORS' CONCLUSIONS: At present, it is uncertain whether the timing of chest radiotherapy as such is important for survival. The optimal integration of chemotherapy and chest radiotherapy in patients with limited-stage small cell lung cancer is unknown. Therefore, further research is needed to establish the most effective combination of radiotherapy and chemotherapy in this disease.

Carcinoma, Small Cell↗

PubMed2004

Verification of treatment parameter transfer by means of electronic portal dosimetry.

Electronic portal imaging devices (EPIDs) are mainly used for patient setup verification during treatment but other geometric properties like block shape and leaf positions are also determined. Electronic portal dosimetry allows dosimetric treatment verification. By combining geometric and dosimetric information, the data transfer between treatment planning system (TPS) and linear accelerator can be verified which in particular is important when this transfer is not carried out electronically. We have developed a pretreatment verification procedure of geometric and dosimetric treatment parameters of a 10 MV photon beam using an EPID. Measurements were performed with a CCD camera-based iView EPID, calibrated to convert a greyscale EPID image into a two-dimensional absolute dose distribution. Central field dose calculations, independent of the TPS, are made to predict dose values at a focus-EPID distance of 157.5 cm. In the same EPID image, the presence of a wedge, its direction, and the field size defined by the collimating jaws were determined. The accuracy of the procedure was determined for open and wedged fields for various field sizes. Ionization chamber measurements were performed to determine the accuracy of the dose values measured with the EPID and calculated by the central field dose calculation. The mean difference between ionization chamber and EPID dose at the center of the fields was 0.8 +/- 1.2% (1 s.d.). Deviations larger than 2.5% were found for half fields and fields with a jaw in overtravel. The mean difference between ionization chamber results and the independent dose calculation was -0.21 +/- 0.6% (1 s.d.). For all wedged fields, the presence of the wedge was detected and the mean difference in actual and measured wedge direction was 0 +/- 3 degrees (1 s.d.). The mean field size differences in X and Y directions were 0.1 +/- 0.1 cm and 0.0 +/- 0.1 cm (1 s.d.), respectively. Pretreatment monitor unit verification is possible with high accuracy and also geometric parameters can be verified using the same EPID image.

Algorithms↗

PubMed2003

Non-invasive 19F MR spectroscopy of 5-fluorocytosine to 5-fluorouracil conversion by recombinant Salmonella in tumours.

The aim of this study was to evaluate the applicability of fluorine-19 magnetic resonance spectroscopy ((19)F MRS) for monitoring in vivo the conversion of 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) after using an attenuated Salmonella Typhimurium strain recombinant to provide cytosine deaminase (TAPET-CD). The (19)F MRS measurements were done on mice bearing the human colon tumour xenograft (HCT116). The intratumoural conversion is greater when TAPET-CD/5-FC is delivered intratumourally (i.tu.) than when TAPET-CD is delivered intravenously (i.v.) and 5-FC intraperitoneally (i.p.). Repeat measurements of the same tumour also yielded important information on the tumour colonization by TAPET-CD through the correlated 5-FC to 5-FU conversion efficacy. The in vivo MRS spectra were confirmed by in vitro (19)F MRS of perchloric acid extracts of the tumour tissue. No 5-FU metabolites were detectable in vivo in the tumours. However, the in vitro measurements revealed, besides 5-FC and 5-FU, the presence of small amounts of catabolites. Finally, spectra obtained in vitro from liver extracts of tumour-bearing mice treated i.tu. with TAPET-CD/5-FC showed no 5-FU and only little amounts of catabolites. Our data illustrate most importantly the potential of (19)F MRS to monitor biologically-based treatments involving cytosine deaminase.

Animals↗

PubMed2003

Probing the specificity of human myeloma proteins with a random peptide phage library.

Human myeloma proteins (HMPs) from 10 patients with multiple myeloma (MM) were used to affinity-select peptides from a random phage-display peptide library. Binding peptides were identified for the 10 analysed antibodies (eight, immunoglobulin G (IgG), and two, immunoglobulin A (IgA)). The specificity of the binding was confirmed by competitive experiments using phages and chemically synthesized peptides. Interestingly, some phage-displayed peptides were immuno-selected with HMPs isolated from different patients. Sequence alignments and homology searches revealed a significant homology with human proteins (e.g. neural cell adhesion proteins) and pathogen-derived proteins (e.g. herpes simplex virus capsid proteins). The selected peptides could be useful as targeting agents for myeloma cells expressing surface immunoglobulins.

Amino Acid Sequence↗

PubMed2003

Tumor-specific gene delivery using genetically engineered bacteria.

The loco-regional control of cancer remains a major contributor to the treatment outcome for many cancer patients prescribed conventional radiotherapy or chemotherapy. Failure of treatment coupled with the realisation that cancer is essentially a genetic disease has led to development of many clinical protocols based on gene therapy. In this review, we will describe an alternative gene delivery system based on the use of non-pathogenic bacteria. Tumor regressions have been reported long ago in patients with bacterially infected tumors, suggesting that bacteria could target tumors and have local anti-tumor effects. The basis of this phenomenon is attributable to the unique properties of the tumor micro-environment. The presence of hypoxic and/or necrotic areas provides a haven for a number of anaerobic bacteria and over the past 60 years, several strains of anaerobic bacteria have been shown to localise within and cause cell lysis of experimental animal tumors. One of the most important strains in that context is Clostridium. Other bacteria have also been implicated in experimental anti-cancer settings. Of these, attenuated Salmonella strains capable of both selective amplification within tumors and expression of effector genes encoding therapeutic proteins are probably the most promising. We will discuss the potential advantages and the pitfalls of this alternative delivery approach. We will emphasize the importance of hypoxia in solid tumors and discuss the potential of radiation-inducible promoters and combined treatment modalities, involving vascular targeting and radiotherapy. We believe that this approach will act in a complementary way to current radiotherapy and chemotherapy treatments of solid tumors.

Animals↗

PubMed2003

Vascular targeting: a potential additional anti-cancer treatment.

Our preclinical in vivo investigations were aimed to evaluate the potential of selectively targeting the tumour vasculature as an additional anti-cancer strategy. Using a clinical angiography method and the tumour growth delay assay, the efficacy of the vascular targeting compound combretastatin A-4 phosphate was demonstrated in rat rhabdomyosarcomas: specifically, an inverse efficacy as compared to radio- or chemotherapy was measured when comparing small and large tumours. The combination of this vascular targeting compound with ionising radiation indicated, depending on the timing and the sequence, a potential benefit. Within the limits of our experiments, no significant increase in tumour growth delay was measured when TNP-470 anti-angiogenesis was given after the combretastatin A-4 phosphate treatment. The use of the vascular targeting agent did advance the in vivo application of a non-apathogenic anaerobe Clostridium transfer system of therapeutic proteins. A strong improvement of the selective expression of cytosine deaminase in the tumour microenvironment was observed, even with very small tumours. In summary, the present preclinical results demonstrate several advantages from the introduction of vascular targeting next to classical and novel anti-cancer therapies.

Angiography, Digital Subtraction↗

PubMed2002

Hypoxia as a target for combined modality treatments.

There is overwhelming evidence that solid human tumours grow within a unique micro-environment. This environment is characterised by an abnormal vasculature, which leads to an insufficient supply of oxygen and nutrients to the tumour cells. These characteristics of the environment limit the effectiveness of both radiotherapy and chemotherapy. Measurement of the oxygenation status of human tumours has unequivocally demonstrated the importance of this parameter on patient prognosis. Tumour hypoxia has been shown to be an independent prognostic indicator of poor outcome in prostate, head and neck and cervical cancers. Recent laboratory and clinical data have shown that hypoxia is also associated with a more malignant phenotype, affecting genomic stability, apoptosis, angiogenesis and metastasis. Several years ago, scientists realised that the unique properties within the tumour micro-environment could provide the basis for tumour-specific therapies. Efforts that are underway to develop therapies that exploit the tumour micro-environment can be categorised into three groups. The first includes agents that exploit the environmental changes that occur within the micro-environment such as hypoxia and reduced pH. This includes bioreductive drugs that are specifically toxic to hypoxic cells, as well as hypoxia-specific gene delivery systems. The second category includes therapies designed to exploit the unique properties of the tumour vasculature and include both angiogenesis inhibitors and vascular targeting agents. The final category includes agents that exploit the molecular and cellular responses to hypoxia. For example, many genes are induced by hypoxia and promoter elements from these genes can be used for the selective expression of therapeutic proteins in hypoxic tumour cells. An overview of the various properties ascribed to tumour hypoxia and the current efforts underway to exploit hypoxia for improving cancer treatment will be discussed.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2002

Clostridium spores for tumor-specific drug delivery.

Insufficient blood supply of rapidly growing tumors leads to the presence of hypoxia, a well-known feature in solid tumors. Hypoxia is known to decrease the efficiency of currently used anti-cancer modalities like surgery, chemotherapy and radiotherapy. Therefore, hypoxia seems to be a major limitation in current anti-cancer therapy. The use of non-pathogenic clostridia to deliver toxic agents to the tumor cells takes advantage of this unique physiology. These strictly anaerobic, Gram-positive, spore-forming bacteria give, after systemic administration, a selective colonization of hypoxic/necrotic areas within the tumor. Moreover, they can be genetically modified to secrete therapeutic proteins like cytosine deaminase or tumor necrosis factor-alpha. The specificity of this protein delivery system can be further increased when expression is controlled by the use of a radio-inducible promoter, leading to increased spatial and temporal regulation of protein expression. This approach of bacterial vector systems to target protein expression to the tumor can be considered very safe since bacteria can be eliminated at any moment by the addition of proper antibiotics. The Clostridium-based delivery system thus presents an alternative therapeutic modality to deliver anti-tumor agents specifically to the tumor site. This high selectivity offers a major advantage in comparison with the classical gene therapy systems.

Animals↗

PubMed2001

Two-stage rotational disordering of a molecular crystal surface: C60.

We propose a two-stage mechanism for the rotational surface disordering phase transition of a molecular crystal, as realized in C60 fullerite. Our study, based on Monte Carlo simulations, uncovers the existence of a new intermediate regime, between a low-temperature ordered (2x2) state, and a high-temperature (1x1) disordered phase. In the intermediate regime there is partial disorder, strongest for a subset of particularly frustrated surface molecules. These concepts and calculations provide a coherent understanding of experimental observations, with possible extension to other molecular crystal surfaces.

Journal Article↗

PubMed2001

Stopping of acoustic waves by sonic polymer-fluid composites.

A two-dimensional periodic array of air cylinders in water is known to have giant acoustic stop bands [M.S. Kushwaha and B. Djafari-Rouhani, J. Appl. Phys. 84 (1998) 4677]. It is shown in the present paper that hollow cylinders made of an elastically-soft polymer containing air inside and arranged on a square lattice in water can still give rise to large acoustic band gaps. Similar properties can also be obtained with a close-packed array of tubes containing water when arranged on a honeycomb lattice in air. The transmission coefficient of films made of such polymer-fluid composites has been calculated by finite difference time domain method. With film thickness not exceeding 75 mm, a deep sonic attenuation band was found with, in the best cases, a lower limit below 1 kHz and an upper limit above 10 kHz.

Journal Article↗

PubMed2001

In vivo antitumor effect of vascular targeting combined with either ionizing radiation or anti-angiogenesis treatment.

PURPOSE: Interference with the tumor blood vessels through anti-angiogenesis or vascular targeting can indirectly suppress tumor growth. Vascular targeting of solid tumors, using tubulin-compromising agents, seems a promising and selective novel treatment. We aimed to evaluate the potential (hypothesis-based) benefit from combinations of vascular targeting using combretastatin A-4 phosphate (combreAp) with either ionizing radiation or anti-angiogenesis. METHODS AND MATERIALS: Rhabdomyosarcoma tumor pieces were inplanted subcutaneously (s.c.) in the lower flank region of syngeneic adult WAG/Rij rats. Tumors were grown until different sizes and stratified for the various treatment groups: small (1-3 cm3), medium (3.1-7 cm3), and large (7.1-14 cm3). CombreAp was injected i.p.; injections of TNP-470 were s.c. in the neck area. Localized single-dose (8 Gy) irradiations of tumors were done under Nembutal anesthesia, always 1 day before a single combreAp (25 mg/kg) injection. The TNP-470 treatment (3 times 30 mg/kg in 1 week) started 1 day after a double (8 days interval between both) combreAp administration. Tumor responses were evaluated by the growth delay assay, and statistical significance of tumor growth change was computed. RESULTS: Large tumors responded better to combreAp treatment given alone than did the smaller ones, confirming our previous data with this tumor model. Combining irradiation with combreAp also resulted in a tumor size-dependent growth delay. With small and medium tumor volumes, a similar response was measured after the combination treatment when compared with irradiation only. Large tumors, however, showed a strong (at least additive) increase of the growth delay with the combined therapy; the difference in tumor growth between the two treatment groups was very significant (p < 0.0001). m When TNP-470 was combined with combreAp, no significant lengthening of the growth delay, irrespective of the tumor size, was present with the applied schedule. CONCLUSION: The current data show a significant advantage in the combination of combreAp with irradiation in rhabdomyosarcomas having a large size (7-14 cm3) at treatment. Such a benefit in tumor response was not observed with the smaller tumors, seemingly because irradiation as such was very effective. No significant gain in growth delay for any tumor size was observed when TNP-470, showing efficacy on its own specifically with tumors measuring <7 cm3, was added to the combreAp treatment. This presumably reflects only little angiogenesis during the first week of rhabdomyosarcoma regrowth after the combreAp treatment.

Angiogenesis Inhibitors↗

PubMed2001

Low-dose hypersensitivity: current status and possible mechanisms.

PURPOSE: To retain cell viability, mammalian cells can increase damage repair in response to excessive radiation-induced injury. The adaptive response to small radiation doses is an example of this induced resistance and has been studied for many years, particularly in human lymphocytes. This review focuses on another manifestation of actively increased resistance that is of potential interest for developing improved radiotherapy, specifically the phenomenon in which cells die from excessive sensitivity to small single doses of ionizing radiation but remain more resistant (per unit dose) to larger single doses. In this paper, we propose possible mechanisms to explain this phenomenon based on our data accumulated over the last decade and a review of the literature. CONCLUSION: Typically, most cell lines exhibit hyper-radiosensitivity (HRS) to very low radiation doses (<10 cGy) that is not predicted by back-extrapolating the cell survival response from higher doses. As the dose is increased above about 30 cGy, there is increased radioresistance (IRR) until at doses beyond about 1 Gy, radioresistance is maximal, and the cell survival follows the usual downward-bending curve with increasing dose. The precise operational and activational mechanism of the process is still unclear, but we propose two hypotheses. The greater amount of injury produced by larger doses either (1) is above a putative damage-sensing threshold for triggering faster or more efficient DNA repair or (2) causes changes in DNA structure or organization that facilitates constitutive repair. In both scenarios, this enhanced repair ability is decreased again on a similar time scale to the rate of removal of DNA damage.

Adaptation, Physiological↗

PubMed2001

BOLD contrast fMRI of whole rodent tumour during air or carbogen breathing using echo-planar imaging at 1.5 T.

The aim of this study was to evaluate the feasibility of functional MR imaging (fMRI) at 1.5 T, exploiting blood oxygenation level-dependent (BOLD) contrast, for detecting changes in whole-tumour oxygenation induced by carbogen (5% CO2+95% O2) inhalation of the host. Adult WAG/Rij rats with rhabdomyosarcomas growing subcutaneously in the lower flank were imaged when tumours reached sizes between 1 and 11 cm3 (n=12). Air and carbogen were alternatively supplied at 2 l/min using a snout mask. Imaging was done on a 1.5-T MR scanner using a T2*-weighted gradient-echo, echo-planar imaging (GE-EPI) sequence. Analysis of the whole-tumour EPI images was based on statistical parametric maps. Voxels with and without signal intensity changes (SIC) were recorded. Significance thresholds were set at p<0.05, corrected for multiple comparisons. In continuous air breathing condition, 3 of 12 tumours showed significant negative SIC and 1 tumour had a clear-cut positive SIC. The remaining tumours showed very little or no change. When switching to carbogen breathing, the SIC were significantly positive in 10 of 12 tumours. Negative SIC were present in 4 tumours, of which three were simultaneously characterised by positive SIC. The overall analysis indicated that 6 of the 12 tumours could be considered as strong positive responders to carbogen. Our research demonstrates the applicability of fMRI GE-EPI at 1.5 T to study whole-tumour oxygenation non-invasively. The observed negative SIC during air condition may reflect the presence of transient hypoxia during these measurements. Selection of tumours on the basis of their individual response to carbogen is possible, indicating a role of such non-invasive measurements for using tailor-made treatments.

Animals↗

PubMed2001

Efficient isolation of total RNA from Clostridium without DNA contamination.

Several molecular techniques require high-quality RNA, free from DNA. Various methods have been described to obtain RNA to be used in expression studies or as starting material in differential display-reverse transcriptase (dd-RT-PCR), for which high-quality RNA free from DNA is an essential requirement. In this report, we compare three different methods to isolate RNA from Gram-positive bacteria: (1) An acid-phenol extraction protocol. (2) The "RNeasy mini kit" from QIAGEN (Valencia, CA, USA). (3) The "SV Total RNA Isolation System" from Promega (Madison, WI, USA).The QIAGEN-kit delivers the highest amount of RNA with the highest purity. Slot blot analysis and dd-RT-PCR confirm the absence of DNA contamination and Northern blot analysis and dd-RT-PCR show high quality of the extracted RNA. This RNA extraction method thus addresses current problems by permitting rapid and safe isolation with high yields of intact RNA for subsequent analysis.

Blotting, Northern↗