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Methods for quantification of in situ hybridization signals obtained by film autoradiography and phosphorimaging applied for estimation of regional levels of calmodulin mRNA classes in the rat brain.

机译:通过胶片放射自显影和磷光成像获得的原位杂交信号的定量方法可用于估算大鼠脑中钙调蛋白mRNA类型的区域水平。

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摘要

Comparative analysis of the regional abundances of the various mRNAs in neural tissues requires the quantitation of target nucleic acid sequences while their tissue distribution is preserved. A quantitative in situ hybridization protocol is presented for the assessment of regional levels of calmodulin (CaM) I, II and III mRNAs in the rat brain. Coronal brain cryostat sections were hybridized with multiple CaM [35S]cRNA probes and co-exposed to an autoradiographic film or storage phosphor screen, together with a membrane-based radioactive standard scale. The membrane scale was calibrated against a brain paste standard scale. Regression analyses of the sensitometric graphs of standard scales corresponding to the autoradiographic film and to the storage phosphor screen were performed by means of exponential (ROD=p(1)(1-exp[-p(2)x])) and linear (LI=ax) functions, respectively (ROD is relative optical density, LI is labeling intensity, and x is radioactivity). The ROD/LI values for the hybridized brain regions were converted into cRNA probe copy numbers (estimations of mRNA copy numbers) through use of the above standard scales. This method was applied to compare the regional abundances of multiple CaM mRNAs in the brains of control, dehydrated, chronic ethanol-treated and ethanol withdrawal-treated animals.
机译:对神经组织中各种mRNA的区域丰度进行比较分析,需要对靶核酸序列进行定量,同时保留其组织分布。提出了定量原位杂交方案,用于评估大鼠脑中钙调蛋白(CaM)I,II和III mRNA的区域水平。将冠状动脉低温恒温器切片与多个CaM [35S] cRNA探针杂交,并与基于膜的放射性标准量表一起共同暴露于放射自显影胶片或荧光粉存储屏幕中。相对于脑膏标准量表校准膜量表。通过指数(ROD = p(1)(1-exp [-p(2)x]))和线性(ROD = p(1)(1-exp [-p(2)x])进行与放射自显影胶片和荧光粉屏幕相对应的标准比例感光度图的回归分析。 LI = ax)分别起作用(ROD是相对光密度,LI是标记强度,x是放射性)。通过使用上述标准量表,将杂交脑区域的ROD / LI值转换为cRNA探针拷贝数(mRNA拷贝数的估计值)。该方法用于比较对照,脱水,慢性乙醇处理和乙醇戒断处理动物大脑中多个CaM mRNA的区域丰度。

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