This is an RSS newsfeed from BioMed Central It is intended to be used with an RSS reader. For more information about RSS newsfeeds from BioMed Central, visit http://www.biomedcentral.com/info/about/rss/ http://www.biomedcentral.com/bmcmolbiol/mostviewed/ Most viewed articles in last 30 days from BMC Molecular Biology (ISSN 1471-2199) published by BioMed Central Background: Gene knock down by RNAi is a highly effective approach to silence gene expression in experimental as well as therapeutic settings. However, this widely used methodology entails serious pitfalls, especially concerning specificity of the RNAi molecules. Results: We tested the most widely used control siRNA directed against GFP for off-target effects and found that it deregulates in addition to GFP a set of endogenous target genes. The off-target effects were dependent on the amount of GFP siRNA transfected and were detected in a variety of cell lines. Since the respective siRNA molecule specific for GFP is widely used as negative control for RNAi experiments, we studied the complete set of off-target genes of this molecule by genome-wide expression profiling. The detected modulated mRNAs had target sequences homologous to the siRNA as small as 8 basepairs in size. However, we found no restriction of sequence homology to 3'UTR of target genes. Conclusion: We can show that even siRNAs without a physiological target have sequence-specific off-target effects in mammalian cells. Furthermore, our analysis defines the off-target genes affected by the siRNA that is commonly used as negative control and directed against GFP. Since off-target effects can hardly be avoided, the best strategy is to identify false positives and exclude them from the results. To this end, we provide the set of false positive genes deregulated by the commonly used GFP siRNA as a reference resource for future siRNA experiments. http://www.biomedcentral.com/1471-2199/9/60 Cordula Tschuch, Angela Schulz, Armin Pscherer, Wiebke Werft, Axel Benner, Agnes Hotz-Wagenblatt, Leticia Serra Barrionuevo, Peter Lichter and Daniel Mertens BMC Molecular Biology 2008, 9:60 Number of accesses: 919 2008-06-24 doi:10.1186/1471-2199-9-60 BMC Molecular Biology 1471-2199 9 60 2008-06-24 Background: As an epigenetic regulator, the transcriptional intermediary factor 1β (TIF1β)/KAP1/TRIM28) has been linked to gene expression and chromatin remodeling at specific loci by association with members of the heterochromatin protein 1 (HP1) family and various other chromatin factors. The interaction between TIF1β and HP1 is crucial for heterochromatin formation and maintenance. The HP1-box, PXVXL, of TIF1β is responsible for its interaction with HP1. However, the underlying mechanism of how the interaction is regulated remains poorly understood. Results: This work demonstrates that TIF1β is phosphorylated on Ser473, the alteration of which is dynamically associated with cell cycle progression and functionally linked to transcriptional regulation. Phosphorylation of TIF1β/Ser473 coincides with the induction of cell cycle gene cyclin A2 at the S-phase. Interestingly, chromatin immunoprecipitation demonstrated that the promoter of cyclin A2 gene is occupied by TIF1β and that such occupancy is inversely correlated with Ser473 phosphorylation. Additionally, when HP1β was co-expressed with TIF1β/S473A, but not TIF1β/S473E, the colocalization of TIF1β/S473A and HP1β to the promoters of Cdc2 and Cdc25A was enhanced. Non-phosphorylated TIF1β/Ser473 allowed greater TIF1β association with the regulatory regions and the consequent repression of these genes. Consistent with possible inhibition of TIF1β's corepressor function, the phosphorylation of the Ser473 residue, which is located near the HP1-interacting PXVXL motif, compromised the formation of TIF1β-HP1 complex. Finally, we found that the phosphorylation of TIF1β/Ser473 is mediated by the PKCδ pathway and is closely linked to cell proliferation. Conclusion: The modulation of HP1β-TIF1β interaction through the phosphorylation/de-phosphorylation of TIF1β/Ser473 may constitute a molecular switch that regulates the expression of particular genes. Higher levels of phosphorylated TIF1β/Ser473 may be associated with the expression of key regulatory genes for cell cycle progression and the proliferation of cells. http://www.biomedcentral.com/1471-2199/9/61 Chiung-Wen Chang, Han-Yi Chou, Yu-Sheng Lin, Kuo-Hsiang Huang, Ching-Jin Chang, Tsui-Chun Hsu and Sheng-Chung Lee BMC Molecular Biology 2008, 9:61 Number of accesses: 701 2008-07-01 doi:10.1186/1471-2199-9-61 BMC Molecular Biology 1471-2199 9 61 2008-07-01 Background: Normalizing to housekeeping gene (HKG) can make results from quantitative real-time PCR (qRT-PCR) more reliable. Recent studies have shown that no single HKG is universal for all experiments. Thus, a suitable HKG should be selected before its use. Only a few studies on HKGs have been done in plants, and none in soybean, an economically important crop. Therefore, the present study was conducted to identify suitable HKG(s) for normalization of gene expression in soybean. Results: All ten HKGs displayed a wide range of Ct values in 21 sample pools, confirming that they were variably expressed. GeNorm was used to determine the expression stability of the HGKs in seven series sets. For all the sample pools analyzed, the stability rank was ELF1B, CYP2 > ACT11 > TUA > ELF1A > UBC2 > ACT2/7 > TUB > G6PD > UBQ10. For different tissues under the same developmental stage, the rank was ELF1B, CYP2 > ACT2/7 > UBC2 > TUA > ELF1A > ACT11 > TUB > G6PD > UBQ10. For the developmental stage series, the stability rank was ACT2/7, TUA > ELF1A > UBC2 > ELF1B > TUB > CYP2 > ACT11 > G6PD > UBQ10. For photoperiodic treatments, the rank was ACT11, ELF1B > CYP2 > TUA > ELF1A > UBC2 > ACT2/7 > TUB > G6PD > UBQ10. For different times of the day, the rank was ELF1A, TUA > ELF1B > G6PD > CYP2 > ACT11 > ACT2/7 > TUB > UBC2 > UBQ10. For different cultivars and leaves on different nodes of the main stem, the ten HKGs' stability did not differ significantly. ΔCt approach and 'Stability index' were also used to analyze the expression stability in all 21 sample pools. Results from ΔCt approach and geNorm indicated that ELF1B and CYP2 were the most stable HKGs, and UBQ10 and G6PD the most variable ones. Results from 'Stability index' analysis were different, with ACT11 and CYP2 being the most stable HKGs, and ELF1A and TUA the most variable ones. Conclusion: Our data suggests that HKGs are expressed variably in soybean. Based on the results from geNorm and ΔCt analysis, ELF1B and CYP2 could be used as internal controls to normalize gene expression in soybean, while UBQ10 and G6PD should be avoided. To achieve accurate results, some conditions may require more than one HKG to be used for normalization. http://www.biomedcentral.com/1471-2199/9/59 Bo Jian, Bin Liu, Yurong Bi, Wensheng Hou, Cunxiang Wu and Tianfu Han BMC Molecular Biology 2008, 9:59 Number of accesses: 579 2008-06-23 doi:10.1186/1471-2199-9-59 BMC Molecular Biology 1471-2199 9 59 2008-06-23 Background: The integrity of RNA molecules is of paramount importance for experiments that try to reflect the snapshot of gene expression at the moment of RNA extraction. Until recently, there has been no reliable standard for estimating the integrity of RNA samples and the ratio of 28S:18S ribosomal RNA, the common measure for this purpose, has been shown to be inconsistent. The advent of microcapillary electrophoretic RNA separation provides the basis for an automated high-throughput approach, in order to estimate the integrity of RNA samples in an unambiguous way. Methods: A method is introduced that automatically selects features from signal measurements and constructs regression models based on a Bayesian learning technique. Feature spaces of different dimensionality are compared in the Bayesian framework, which allows selecting a final feature combination corresponding to models with high posterior probability. Results: This approach is applied to a large collection of electrophoretic RNA measurements recorded with an Agilent 2100 bioanalyzer to extract an algorithm that describes RNA integrity. The resulting algorithm is a user-independent, automated and reliable procedure for standardization of RNA quality control that allows the calculation of an RNA integrity number (RIN). Conclusion: Our results show the importance of taking characteristics of several regions of the recorded electropherogram into account in order to get a robust and reliable prediction of RNA integrity, especially if compared to traditional methods. http://www.biomedcentral.com/1471-2199/7/3 Andreas Schroeder, Odilo Mueller, Susanne Stocker, Ruediger Salowsky, Michael Leiber, Marcus Gassmann, Samar Lightfoot, Wolfram Menzel, Martin Granzow and Thomas Ragg BMC Molecular Biology 2006, 7:3 Number of accesses: 427 2006-01-31 doi:10.1186/1471-2199-7-3 BMC Molecular Biology 1471-2199 7 3 2006-01-31 Background: Traumatic brain injury models are widely studied, especially through gene expression, either to further understand implied biological mechanisms or to assess the efficiency of potential therapies. A large number of biological pathways are affected in brain trauma models, whose elucidation might greatly benefit from transcriptomic studies. However the suitability of reference genes needed for quantitative RT-PCR experiments is missing for these models. Results: We have compared five potential reference genes as well as total cDNA level monitored using Oligreen reagent in order to determine the best normalizing factors for quantitative RT-PCR expression studies in the early phase (0- 48h post-trauma (PT)) of a murine model of diffuse brain injury. The levels of 18S rRNA, and of transcripts of Beta-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), Beta-microtubulin and S100-Beta were determined in the injured brain region of traumatized mice sacrificed at 30 min, 3h, 6h, 12h, 24h and 48h post-trauma. The stability of the reference genes candidates and of total cDNA was evaluated by three different methods, leading to the following rankings as normalization factors, from the most suitable to the less : by using geNorm VBA applet, we obtained the following sequence : cDNA(Oligreen) ; GAPDH > 18S rRNA > S100-Beta > Beta-microtubulin > Beta-actin; by using NormFinder Excel Spreadsheet, we obtained the following sequence : GAPDH > cDNA(Oligreen) > S100-Beta > 18S rRNA > Beta-actin > Beta-microtubulin; by using a Confidence-Interval calculation, we obtained the following sequence : cDNA(Oligreen) > 18S rRNA ; GAPDH > S100-Beta > Beta-microtubulin > Beta-actin. Conclusion: This work suggests that Oligreen cDNA measurements, 18S rRNA and GAPDH or a combination of them may be used to efficiently normalize qRT-PCR gene expression in mouse brain trauma injury, and that Beta-actin and Beta-microtubulin should be avoided. The potential of total cDNA as measured by Oligreen as a first-intention normalizing factor with a broad field of applications is highlighted. Pros and cons of the three methods of normalization factors selection are discussed. A generic time- and cost-effective procedure for normalization factor validation is proposed. http://www.biomedcentral.com/1471-2199/9/62 Herve Rhinn, Catherine Marchand-Leroux, Nicole Croci, Michel Plotkine, Daniel Scherman and Virginie Escriou BMC Molecular Biology 2008, 9:62 Number of accesses: 416 2008-07-08 doi:10.1186/1471-2199-9-62 BMC Molecular Biology 1471-2199 9 62 2008-07-08 Background: Gene silencing by RNA interference (RNAi) is a powerful tool for functional genomics. Although RNAi was first described in Caenorhabditis elegans, several nematode species are unable to mount an RNAi response when exposed to exogenous double stranded RNA (dsRNA). These include the satellite model organisms Pristionchus pacificus and Oscheius tipulae. Available data also suggest that the RNAi pathway targeting exogenous dsRNA may not be fully functional in some animal parasitic nematodes. The genus Panagrolaimus contains bacterial feeding nematodes which occupy a diversity of niches ranging from polar, temperate and semi-arid soils to terrestrial mosses. Thus many Panagrolaimus species are adapted to tolerate freezing and desiccation and are excellent systems to study the molecular basis of environmental stress tolerance. We investigated whether Panagrolaimus is susceptible to RNAi to determine whether this nematode could be used in large scale RNAi studies in functional genomics. Results: We studied two species: Panagrolaimus sp. PS1159 and Panagrolaimus superbus. Both nematode species displayed embryonic lethal RNAi phenotypes following ingestion of Escherichia coli expressing dsRNA for the C. elegans embryonic lethal genes Ce-lmn-1 and Ce-ran-4. Embryonic lethal RNAi phenotypes were also obtained in both species upon ingestion of dsRNA for the Panagrolaimus genes ef1b and rps-2. Single nematode RT-PCR showed that a significant reduction in mRNA transcript levels occurred for the target ef1b and rps-2 genes in RNAi treated Panagrolaimus sp. 1159 nematodes. Visible RNAi phenotypes were also observed when P. superbus was exposed to dsRNA for structural genes encoding contractile proteins. All RNAi phenotypes were highly penetrant, particularly in P. superbus. Conclusion: This demonstration that Panagrolaimus is amenable to RNAi by feeding will allow the development of high throughput methods of RNAi screening for P. superbus. This greatly enhances the utility of this nematode as a model system for the study of the molecular biology of anhydrobiosis and cryobiosis and as a possible satellite model nematode for comparative and functional genomics. Our data also identify another nematode infraorder which is amenable to RNAi and provide additional information on the diversity of RNAi phenotypes in nematodes. http://www.biomedcentral.com/1471-2199/9/58 Adam J Shannon, Trevor Tyson, Ilona Dix, Jacqueline Boyd and Ann M Burnell BMC Molecular Biology 2008, 9:58 Number of accesses: 407 2008-06-19 doi:10.1186/1471-2199-9-58 BMC Molecular Biology 1471-2199 9 58 2008-06-19 Background: Gene expression has a strong stochastic element resulting in highly variable mRNA levels between individual cells, even in a seemingly homogeneous cell population. Access to fundamental information about cellular mechanisms, such as correlated gene expression, motivates measurements of multiple genes in individual cells. Quantitative reverse transcription PCR (RT-qPCR) is the most accessible method which provides sufficiently accurate measurements of mRNA in single cells. Results: Low concentration of guanidine thiocyanate was used to fully lyse single pancreatic β-cells followed by RT-qPCR without the need for purification. The accuracy of the measurements was determined by a quantitative noise-model of the reverse transcription and PCR. The noise is insignificant for initial copy numbers >100 while at lower copy numbers the noise intrinsic of the PCR increases sharply, eventually obscuring quantitative measurements. Importantly, the model allows us to determine the RT efficiency without using artificial RNA as a standard. The experimental setup was applied on single endocrine cells, where the technical and biological noise levels were determined. Conclusion: Noise in single-cell RT-qPCR is insignificant compared to biological cell-to-cell variation in mRNA levels for medium and high abundance transcripts. To minimize the technical noise in single-cell RT-qPCR, the mRNA should be analyzed with a single RT reaction, and a single qPCR reaction per gene. http://www.biomedcentral.com/1471-2199/9/63 Martin Bengtsson, Martin Hemberg, Patrik Rorsman and Anders Ståhlberg BMC Molecular Biology 2008, 9:63 Number of accesses: 390 2008-07-17 doi:10.1186/1471-2199-9-63 BMC Molecular Biology 1471-2199 9 63 2008-07-17 Background: Control genes, which are often referred to as housekeeping genes, are frequently used to normalise mRNA levels between different samples. However, the expression level of these genes may vary among tissues or cells and may change under certain circumstances. Thus, the selection of housekeeping genes is critical for gene expression studies. To address this issue, 7 candidate housekeeping genes including several commonly used ones were investigated in isolated human reticulocytes. For this, a simple ΔCt approach was employed by comparing relative expression of 'pairs of genes' within each sample. On this basis, stability of the candidate housekeeping genes was ranked according to repeatability of the gene expression differences among 31 samples. Results: Initial screening of the expression pattern demonstrated that 1 of the 7 genes was expressed at very low levels in reticulocytes and was excluded from further analysis. The range of expression stability of the other 6 genes was (from most stable to least stable): GAPDH (glyceraldehyde 3-phosphate dehydrogenase), SDHA (succinate dehydrogenase), HPRT1 (hypoxanthine phosphoribosyl transferase 1), HBS1L (HBS1-like protein) and AHSP (alpha haemoglobin stabilising protein), followed by B2M (beta-2-microglobulin). Conclusion: Using this simple approach, GAPDH was found to be the most suitable housekeeping gene for expression studies in reticulocytes while the commonly used B2M should be avoided. http://www.biomedcentral.com/1471-2199/7/33 Nicholas Silver, Steve Best, Jie Jiang and Swee Lay Thein BMC Molecular Biology 2006, 7:33 Number of accesses: 367 2006-10-06 doi:10.1186/1471-2199-7-33 BMC Molecular Biology 1471-2199 7 33 2006-10-06 Background: Housekeeping genes are commonly used as endogenous reference genes for the relative quantification of target genes in gene expression studies. No conclusive systematic study comparing the suitability of different candidate reference genes in clear cell renal cell carcinoma has been published to date. To remedy this situation, 10 housekeeping genes for normalizing purposes of RT-PCR measurements already recommended in various studies were examined with regard to their usefulness as reference genes. Results: The expression of the potential reference genes was examined in matched malignant and non-malignant tissue specimens from 25 patients with clear cell renal cell carcinoma. Quality assessment of isolated RNA performed with a 2100 Agilent Bioanalyzer showed a mean RNA integrity number of 8.7 for all samples. The between-run variations related to the crossing points of PCR reactions of a control material ranged from 0.17% to 0.38%. The expression of all genes did not depend on age, sex, and tumour stage. Except the genes TATA box binding protein (TBP) and peptidylprolyl isomerase A (PPIA), all genes showed significant differences in expression between malignant and non-malignant pairs. The expression stability of the candidate reference genes was additionally controlled using the software programs geNorm and NormFinder. TBP and PPIA were validated as suitable reference genes by normalizing the target gene ADAM9 using these two most stably expressed genes in comparison with up- and down-regulated housekeeping genes of the panel. Conclusion: Our study demonstrated the suitability of the two housekeeping genes PPIA and TBP as endogenous reference genes when comparing malignant tissue samples with adjacent normal tissue samples from clear cell renal cell carcinoma. Both genes are recommended as reference genes for relative gene quantification in gene profiling studies either as single gene or preferably in combination. http://www.biomedcentral.com/1471-2199/8/47 Monika Jung, Azizbek Ramankulov, Jan Roigas, Manfred Johannsen, Martin Ringsdorf, Glen Kristiansen and Klaus Jung BMC Molecular Biology 2007, 8:47 Number of accesses: 365 2007-06-08 doi:10.1186/1471-2199-8-47 BMC Molecular Biology 1471-2199 8 47 2007-06-08 Background: Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule. Results: We describe a real-time PCR technique employing attached universal duplex probes (AUDP), which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP) and a complementary quenching probe (QP) lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT) and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO) quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods. Conclusion: The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost. http://www.biomedcentral.com/1471-2199/9/54 Litao Yang, Wanqi Liang, Lingxi Jiang, Wenquan Li, Wei Cao, Zoe A Wilson and Dabing Zhang BMC Molecular Biology 2008, 9:54 Number of accesses: 349 2008-06-04 doi:10.1186/1471-2199-9-54 BMC Molecular Biology 1471-2199 9 54 2008-06-04