共查询到20条相似文献,搜索用时 15 毫秒
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探讨显微切割过程中有效保持RNA完整性的组织固定方法,建立一种简易的手工显微切割法.应用自制“T形板”辅助冰冻切片,100%无水乙醇一次性脱水固定,“排除切割法”获取目的细胞,用TRIzol提取RNA,琼脂糖凝胶电泳和RT-PCR分析RNA质量.“一步法”固定可长时间保存RNA的完整性;从食管癌标本5个特定阶段的细胞中提取的RNA,经电泳和RT-PCR分析均具有较高的质量.无水乙醇“一步法”固定,在显微切割的过程中可有效保持RNA的完整性;T形板和“排除切割法”简化了手工显微切割的操作,提取的RNA质、量均可满足后续分子水平研究的需要. 相似文献
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通过RT-PCR技术扩增了甲肝病毒减毒株(H2)全长RNA,并对长片段RT-PCR扩增进行了方法学上的探讨.采用抗血清特异沉淀病毒;盐酸胍-酸性酚、氯仿一步法分离纯化病毒RNA,可得到高质量的RNA样品;以此RNA为模板,在无RNA酶的逆转录酶作用下,合成单链cDNA;继续以此cDNA为模板,利用32 mer寡核苷酸引物, 在Taq和Deep Vent DNA多聚酶的作用下进行PCR扩增,得到7.4 kb的扩增产物. 相似文献
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Shan Z Lin Q Deng C Li X Huang W Tan H Fu Y Yang M Yu XY 《Molecular biology reports》2009,36(6):1483-1489
Gene silencing can be mediated by small interfering RNA (siRNA) and microRNA (miRNA). To investigate the potential application
of using a precursor microRNA (pre-miRNA) backbone for gene silencing, we studied the inhibition efficiency of exogenous GFP
and endogenous GAPDH by conventional shRNA- and pre-miRNA-designed hairpins, respectively. In this study, the conventional
shRNA-, pre-miRNA-30-, and pre-miRNA-155-designed hairpins targeting either GFP or GAPDH were transfected into the HEK293
cells that were mediated by the pSilencer-4.1-neo vector, which carries a modified RNA polymerase II-type CMV promoter. Comparisons
with conventional GFP shRNA showed that GFP levels were reduced markedly by pre-miRNA-30- and pre-miRNA-155-designed GFP shRNAs
by fluorescence microscopy. The consistent results from semi-quantitative RT-PCR and Western blot analysis revealed that pre-miRNA-30-
and pre-miRNA-155-designed GFP shRNAs could suppress GFP expression significantly. As for endogenous GAPDH, the results from
semi-quantitative RT-PCR and Western blot analysis showed that pre-miRNA-30- and pre-miRNA-155-designed GAPDH shRNAs could
suppress GAPDH expression even more efficiently than conventional GAPDH shRNA. Together, this study confirmed the efficiency
of gene silencing mediated by pre-miRNA-30- and pre-miRNA-155-designed shRNAs, demonstrating that pre-miRNA-designed hairpins
are a good strategy for gene silencing. 相似文献
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利用RT-PCR技术扩增了口蹄疫病毒(FMDV)编码RNA依赖的RNA聚合酶的3D基因,并将其克隆到原核表达质粒载体pET-28a( )中。3D基因经测序确认后在大肠杆菌BL-21中表达,表达产物纯化的目的蛋白进行Western-blotting检测,获得分子量约55KDa的单一3D基因表达产物。利用RNA体外复制体系和荧光定量PCR技术,证明纯化的3D基因表达产物RNA依赖的RNA聚合酶具有较高的酶活性,可以在体外从头合成FMDVRNA,且主要以引物依赖的方式合成病毒基因组。 相似文献
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Nasser Al-Shanti Amarjit Saini Claire E. Stewart 《Journal of biomolecular techniques》2009,20(3):172-179
Quantitative RT-PCR can be carried out as a one- or a two-step reaction. However, the choice of method raises controversy from the perspective of the researcher and manufacturer, because of advantages and disadvantages with both systems. We therefore hypothesize that running the RNA-to-CT™ 2-Step kit [(Applied Biosystems (AB), Foster City, CA] using a one-step protocol (as recommended) is not appropriate for quantitation of gene expression levels and should not be performed. Consequently, we ran comparative studies of the two suggested methods to evaluate their efficiency, sensitivity, and accuracy. To ensure precession, two different PCR machines were used: the StepOnePlus system and Chromo4. In addition, the RNA-to-CT™ 1-Step kit (recently launched by AB) was also used to compare its efficiency with these methods. Efficiency, sensitivity, and linearity were determined by standard curves generated using RNA isolated from C2 myoblasts to amplify the housekeeping gene GAPDH. When the RNA-to-CT™ 2-Step kit was run as a two-step reaction on the Chromo4 or StepOnePlus, respectively, not only did the efficiency increase (100±1.5% and 99.7±0.95%) but also the sensitivity (comparative threshold cycle for the lowest standard: 33.2±0.5 and 32.5±0.7) and linearity (0.997±0.001 and 0.993±0.006) compared with RNA-to-CT™ 2-Step run as one-step and RNA-to-CT™ 1-Step kit. This is the first study to demonstrate that the RNA-to-CT™ 2-Step kit is not reliable to be performed as a one-step reaction but as a two-step reaction, is even more sensitive than the newly launched RNA-to-CT™ 1-Step kit. 相似文献
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Representative cDNA libraries from few plant cells 总被引:22,自引:3,他引:19
Thomas Dresselhaus Horst Lörz Erhard Kranz 《The Plant journal : for cell and molecular biology》1994,5(4):605-610