A-769662
BackgroundAMP-activated protein kinases (AMPK) are a family of protein kinases that are directly activated in response to increased cellular levels of AMP and are thus able to act as a sensor for lowered cellular energy levels. In response to low cellular energy levels, the activation of AMPK causes an increase in cellular levels of ATP by increasing metabolism of glucose and fatty acids.
Activation of AMPK is of clinical importance, as metformin, a biguanide drug (used to treat type II diabetes), exerts its main therapeutic effect by activating AMPK in the liver. However, this is through an indirect mechanism and does not occur in other tissues (probably because metformin is not readily taken up in other cell types). In experimental studies, the cell-permeable drug AICAR-riboside has been used to activate AMPK. AICAR-riboside is readily taken up and metabolised to ZMP, which in turn can activate AMPK. However, ZMP also activates a range of other AMP-activated enzymes, including glycogen phosphorylase and fructose 1:6 bisphosphatase. A-769662 is a cell permeable drug, that potently activates AMPK in cells (EC50 120 nM) and, unlike AICAR-riboside , does not activate other AMP target enzymes [1, 2]. When tested against a panel of 76 protein kinases, A-769662 was observed to only significantly inhibit the activity of PIM1 and PIM3 (>50% inhibition).
References
1 Cool, B., Zinker, B., Chiou, W., Kifle, L., Cao, N., Perham, M., Dickinson, R., Adler, A., Gagne, G., Iyengar, R., Zhao, G., Marsh, K., Kym, P., Jung, P., Camp, H. S. and Frevert, E. (2006) Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome.Cell Metab 3, 403-16
2 Sanders, M. J., Ali, Z. S., Hegarty, B. D., Heath, R., Snowden, M. A. and Carling, D. (2007) Defining the mechanism of activation of AMP-activated protein kinase by the small molecule A-769662, a member of the thienopyridone family.J Biol Chem 282, 32539-48
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引物,又名引子。是一小段单链DNA或RNA,作为DNA复制的起始点,在核酸合成反应时,作为每个多核苷酸链进行延伸的出发点而起作用的多核苷酸链,之所以需要引物是因为在DNA合成中DNA聚合酶仅仅可以把新的核苷酸加到已有的DNA链上。
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上面较高的是样本曲线,下面较低的是未加样本(茎环引物和引物、探针都加入)
一般来讲,进行real-time qPCR MasterMix都是2×的浓缩液,只需要加入模板和引物就可以。由于real-time qPCR灵敏度高,所以每个样品至少要做3个平行孔,以防在后面的数据分析中,由于Ct相差较多或者SD太大,无法进行统计分析。通常来讲,反应体系的引 物终浓度为100-400mM;模板如果是总RNA一般是10ng-500,如果cDNA,通常情况下是1ul或者1ul的10倍稀释液,要根据目的基因 的表达丰度进行调整。当然这些都是经验值,在操作过程中,还需要根据所用MasterMix,模板和引物的不同进行优化,达到一个最佳反应体系。在反应体 系配置过程中,有下面几点需要注意:
1. MasterMix不要反复冻融,如果经常使用,最好溶解后放在4度。
2. 更多的配制Mix进行,减少加样误差。最好能在冰上操作。
3. 每管或每孔都要换新枪头!不要连续用同一个枪头加样!
4. 所有成分加完后,离心去除气泡。
5. 每个样品至少3个平行孔。
设计方法:通用茎环后端加miRNA后面6个碱基的反向互补序列为反转录引物,正向引物为miRNA去除后面6个碱基的序列,反向引物在茎环上。
反转录茎环通用序列:
GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGAC
miRNA序列:
>mmu-miR-99b-5p
MIMAT0000132 Mus musculus miR-99b-5p
CACCCGUAGAACCGACCUUGCG
mmu-miR-99b-5p反转录颈环引物为:
GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCGCAAG
定量PCR反向引物为:TGTCGTGGAGTCGGC
正向引物为:CACCCGTAGAACCGAC
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