Adenosine triphosphate (ATP) is the most abundant and primary carrier of the required energy for various functions in cells. Prolonged ischemia, reperfusion, anaerobic metabolism and lactate accumulation can lead to a dramatic decrease of ATP, cell swelling, cell rupture, and finally cell death by necrotic, necroptotic, apoptotic, and autophagic mechanisms. Due to drastic hydrolysis of ATP in vivo by ectoenzymes and poor cellular penetration, the direct delivery of ATP to the ischemic tissues is difficult.
To increase delivery of ATP to the tissues and protect from enzymatic degradation, encapsulation in liposomes has been proposed and demonstrated in various models of ischemia [1,2]. Studies on myocardial [1,3,4], liver [5-8], retina [9] and wound healing [10-12] ischemia have shown the ability of liposomal encapsulated ATP to prevent cell death and tissue dysfunction following ischemic events.
The encapsulation of ATP in liposomes markedly promotes its effectiveness by preventing the hydrolysis by extracellular enzymes, increasing ATP circulation time and enhancing its intracellular penetration. Depending on the type of the cell line and the target organ various types of liposomes with different surface charges such as anionic, cationic and neutral has been studied by various groups. Moreover, ATP encapsulated liposomes has been demonstrated to improve energy state and function of the cold-stored liver [6,7,13].
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逆转录(reverse transcription)是以RNA为模板合成DNA的过程,即RNA指导下的DNA合成。此过程中,核酸合成与转录(DNA到RNA)过程与遗传信息的流动方向(RNA到DNA)相反,故称为逆转录。逆转录过程是RNA病毒的复制形式之一,需逆转录酶的催化。 逆转录过程的揭示是分子生物学研究中的重大发现,是对中心法则的重要修正和补充。人们通过体外模拟该过程,以样本中提取的mRNA为模板,在逆转录酶的作用下,合成出互补的cDNA,构建cDNA文库,并从中筛选特异的目的基因。该方法已成为基因工程技术中最常用的获得目的基因的策略之一。
1.模板提取(一般为RNA):Trizol、氯仿、异丙醇、无水乙醇、DEPC处理水
2.模板浓度测定:分光光度计或NanoDrop
3.逆转录:逆转录试剂盒(或者一步法试剂盒),这一步可以用普通PCR做,也可以用水域做。
4.荧光定量PCR试剂:通常有用SYBR Green Mix做的,但是这里建议你用EvaGreen做,灵敏度和平行性都要好于SYBR Green,并且如果你那是ABI或者Stratagene的PCR如果用SYBR Green还需要加一步Rox很麻烦。
5.其他:除了以上的那些还需要离心管、PCR管或板(Axygen反应比较好)、移液枪等,暂时就想到这么多。
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大家miRNA逆转录和qPCR的试剂盒用的是那个公司的啊,求推荐!

