The importance of RT-qPCR primer design for the detection of siRNA-mediated mRNA silencing

<p>Abstract</p> <p>Background</p> <p>The use of RNAi to analyse gene function <it>in vitro </it>is now widely applied in biological research. However, several difficulties are associated with its use <it>in vivo</it>, mainly relating to inefficient delivery and non-specific effects of short RNA duplexes in animal models. The latter can lead to false positive results when real-time RT-qPCR alone is used to measure target mRNA knockdown.</p> <p>Findings</p> <p>We observed that detection of an apparent siRNA-mediated knockdown <it>in vivo </it>was dependent on the primers used for real-time RT-qPCR measurement of the target mRNA. Two siRNAs specific for <it>RRM1 </it>with equivalent activity <it>in vitro </it>were administered to A549 xenografts via intratumoural injection. In each case, apparent knockdown of <it>RRM1 </it>mRNA was observed only when the primer pair used in RT-qPCR flanked the siRNA cleavage site. This false-positive result was found to result from co-purified siRNA interfering with both reverse transcription and qPCR.</p> <p>Conclusions</p> <p>Our data suggest that using primers flanking the siRNA-mediated cleavage site in RT-qPCR-based measurements of mRNA knockdown <it>in vivo </it>can lead to false positive results. This is particularly relevant where high concentrations of siRNA are introduced, particularly via intratumoural injection, as the siRNA may be co-purified with the RNA and interfere with downstream enzymatic steps. Based on these results, using primers flanking the siRNA target site should be avoided when measuring knockdown of target mRNA by real-time RT-qPCR.</p

A rapid and sensitive method to detect siRNA-mediated mRNA cleavage in vivo using 5′ RACE and a molecular beacon probe

Specific detection of mRNA cleavage by 5′RACE is the only method to confirm the knockdown of mRNA by RNA interference, but is rarely reported for in vivo studies. We have combined 5′-RNA-linker-mediated RACE (5′-RLM-RACE) with real-time PCR using a molecular beacon to develop a rapid and specific method termed MBRACE, which we have used to detect small-interfering RNA (siRNA)-induced cleavage of ApoB, RRM1 and YBX1 transcripts in vitro, and ApoB in vivo. When RNA from siRNA-transfected cells was used for 5′-RLM-RACE and a cleavage site-specific molecular beacon probe was included in subsequent real-time PCR analysis, the specific mRNA cleavage product was detected. Detection of siRNA-mediated cleavage was also observed when RNA from mouse liver following administration of ApoB-specific siRNA was analysed, even in cases where ApoB knockdown measured by real-time PCR was <10%. With its sensitivity and specificity, this variation on the 5′RACE method should prove a useful tool to detect mRNA cleavage and corroborate knockdown studies following siRNA use in vivo

Relevance of Translation Initiation in Diffuse Glioma Biology and its Therapeutic Potential

Cancer cells are continually exposed to environmental stressors forcing them to adapt their protein production to survive. The translational machinery can be recruited by malignant cells to synthesize proteins required to promote their survival, even in times of high physiological and pathological stress. This phenomenon has been described in several cancers including in gliomas. Abnormal regulation of translation has encouraged the development of new therapeutics targeting the protein synthesis pathway. This approach could be meaningful for glioma given the fact that the median survival following diagnosis of the highest grade of glioma remains short despite current therapy. The identification of new targets for the development of novel therapeutics is therefore needed in order to improve this devastating overall survival rate. This review discusses current literature on translation in gliomas with a focus on the initiation step covering both the cap-dependent and cap-independent modes of initiation. The different translation initiation protagonists will be described in normal conditions and then in gliomas. In addition, their gene expression in gliomas will systematically be examined using two freely available datasets. Finally, we will discuss different pathways regulating translation initiation and current drugs targeting the translational machinery and their potential for the treatment of gliomas

A rapid and sensitive method to detect siRNA-mediated mRNA cleavage in vivo using 5 0 RACE and a molecular beacon probe

ABSTRACT Specific detection of mRNA cleavage by 5 0 RACE is the only method to confirm the knockdown of mRNA by RNA interference, but is rarely reported for in vivo studies. We have combined 5 0 -RNA-linker-mediated RACE (5 0 -RLM-RACE) with real-time PCR using a molecular beacon to develop a rapid and specific method termed MBRACE, which we have used to detect smallinterfering RNA (siRNA)-induced cleavage of ApoB, RRM1 and YBX1 transcripts in vitro, and ApoB in vivo. When RNA from siRNA-transfected cells was used for 5 0 -RLM-RACE and a cleavage sitespecific molecular beacon probe was included in subsequent real-time PCR analysis, the specific mRNA cleavage product was detected. Detection of siRNA-mediated cleavage was also observed when RNA from mouse liver following administration of ApoB-specific siRNA was analysed, even in cases where ApoB knockdown measured by real-time PCR was &lt;10%. With its sensitivity and specificity, this variation on the 5 0 RACE method should prove a useful tool to detect mRNA cleavage and corroborate knockdown studies following siRNA use in vivo

Study of Strawberry Notch homolog 1 and 2 expression in human glioblastoma.

peer reviewed[en] PURPOSE: In this work, we aimed to comprehensively document the expression of Strawberry Notch homolog (SBNO) 1 and 2 in glioblastoma (GBM) tissue and patient-derived GBM stem-like cell (GSC) cultures. METHODS: We investigated SBNO1 and SBNO2 expression at the RNA and protein levels in glioma patient tissue and GSCs, respectively by performing immunostainings and qPCR analyses. We also used publicly-available datasets for assessing SBNO1 and SBNO2 gene expression and related copy number alterations. We used lentiviral transduction of SBNO2 to analyze the effect of its expression in patient-derived GSCs. RESULTS: We observed that SBNO2 expression is increased in GBM tissue samples compared to non tumoral brain, or lower-grade gliomas, whereas SBNO1 expression remains unchanged. We hypothesized that such SBNO2 high expression might be linked to copy-number alterations at the level of the 19p13 chromosome section. We located SBNO1 and SBNO2 in different subcellular compartments. Finally, we observed that SBNO2 overexpression induces different phenotypes in different patient-derived GSCs. CONCLUSION: These results provide the first characterization of SBNO1 and SBNO2 expression in glioma tissue, and indicate SBNO2 as highly expressed in GBM

Additional file 7: Figure S7. of Interferon-γ blocks signalling through PDGFRβ in human brain pericytes

(A, B) Pericytes were treated for four consecutive days (once every 24 h) with either vehicle (Veh), TNFα (5 ng/mL), or IL-1β (1 ng/mL). After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure PDGFRβ and αSMA expression by immunocytochemistry. Quantification of PDGFRβ (A) and αSMA (B) staining, mean ± s.e.m. (n = 3), ****(p < 0.0001), ***(p < 0.001), *(p < 0.05) (two-way ANOVA). (C) Pericytes were treated for three or four consecutive days (once every 24 h) with either vehicle (Veh), TNFα (5 ng/mL), or IL-1β (1 ng/mL). After 48 h, cells were treated with PDGF-BB (10 ng/mL) for either 24 or 48 h. Western blot band intensity of PDGFRβ, αSMA, and GAPDH were quantified, normalized to GAPDH, and plotted as mean ± s.e.m. (n = 3), and differences were not significant (two-way ANOVA). (TIF 1163 kb

Additional file 6: Figure S6. of Interferon-γ blocks signalling through PDGFRβ in human brain pericytes

Pericytes were treated for four consecutive days (once every 24 h) with either vehicle (Veh), TNFα (5 ng/mL), or IL-1β (1 ng/mL). After 48 h of cytokine treatment, cells were treated with either vehicle or PDGF-BB (10 ng/mL) to measure the PDGF-BB-induced proliferative response (A, B). This was done in two ways: after 96 h total treatment, cells were fixed, labelled with a Ki67 antibody and Hoechst (A, C); alternatively, EdU was added to measure cell proliferation over the final 24 h of the experiment (B, C). Positive cells of the total cells measured by Hoechst were quantified and plotted as mean ± s.e.m. (n = 3), ****(p < 0.0001), ***(p < 0.001), *(p < 0.05) from a two-way ANOVA. (TIF 1034 kb