Real-time RT-PCR is made easy with the One Step PrimeScript RT-PCR Kit (Perfect Real Time), a one-step real-time RT-PCR kit designed for real-time RT-PCR (qPCR)via probe-based qPCR assays such as 5" nuclease assays1. In this5" nuclease assay real-time PCR (qPCR) kit, one-step real-time RT-PCR can be performed in a single tube, simplifyingamplification and minimizing the risk of contamination. Amplified products are monitored in real time, so there is no need to verify them through electrophoresisfollowing PCR. The sensitivity provided by the enzymes in this kit enables detection of very small amounts of RNA, including RNA derived from viral, rare, or precioussamples.
Real-time RT-PCR is made easy with the One Step PrimeScript RT-PCR Kit (Perfect Real Time), a one-step real-time RT-PCR kit designed for real-time RT-PCR (qPCR)via probe-based qPCR assays such as 5" nuclease assays1. In this5" nuclease assay real-time PCR (qPCR) kit, one-step real-time RT-PCR can be performed in a single tube, simplifyingamplification and minimizing the risk of contamination. Amplified products are monitored in real time, so there is no need to verify them through electrophoresisfollowing PCR. The sensitivity provided by the enzymes in this kit enables detection of very small amounts of RNA, including RNA derived from viral, rare, or precioussamples.
This kit uses PrimeScript RTase,a modified, recombinant MMLV (Moloney Murine Leukemia Virus) reverse transcriptasethat is verified to have low levels of RNase H and which exhibits an excellent extension rate andquick, full-length cDNA synthesis, as well as Takara Ex Taq HS, ahigh efficiency hot start PCR enzyme optimized for this one-step real-time RT-PCR kit.
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TUNEL的缺点是：1），操作要求高，组织样本需要固定（即使是培养细胞，也需要固定），不恰当的固定方法对实验结果影响很大，导致背景过高或者信号过弱，因此实验结果重复性不好;有的人花上半年做一个体内的TUNEL是一点都不奇怪的。2），大部分诱导凋亡的药物也引起DNA损伤，从而产生DNA断裂，易引入假阳性;3）凋亡晚期细胞基因组大量降解，导致TUNEL标记反而减少，因此此法反应的是早期凋亡比例，不能严格反应凋亡比例，属于半定量研究。
尽管有如此多缺点，但由于其是目前为数不多的能原位标记凋亡细胞的方法，因此用于组织体内凋亡研究仍然是首选。但体外细胞实验研究，很少用此法。
凋亡检测中，TUNEL并不是过时的方法，现在研究凋亡的文献仍然常见。而且相反，还比以前多一点，因为现在体内实验越来越多了，甚至线虫的凋亡研究，都用TUNEL。
凋亡研究中，的确有几种方法过时了，当然是因为有替代方案了，比如DNAladder，电镜。至于AnnexinV是不能用来和TUNEL一起评论的，前者用于细胞，而且主要是悬浮细胞，后者主要用于组织。虽然有人也做镜下的AnnexinV观察，TUNEL的细胞staining，但这都不是主流。
回到lz的原帖，的确如大家所言，做贴壁细胞，不推荐用TUNEL。如果是经典凋亡途径，只是确定比例，用最经典，最常用的subG1法即可，如果是不确定是否是凋亡，用AnnexinV，不过贴壁细胞用此法，要注意消化时间。
那为什么SFDA不批准CA199CEAAFP等检测试剂盒作为癌症检测的手段呢？
而放射性核素标记，是对体外诊断试剂的某些元素进行放射性特征性标记，便于检测而已，这类的放射性强度不大，危害不高
在弱碱性（pH 8~9）、暗处、室温或40℃条件下，氨基酸的α-氨基很容易与2,4-二硝基氟苯（缩写为FDNB或DNFB）反应，生成黄色的2,4-二硝基苯氨基酸（dinitrophenyl amino acid，简称DNP-氨基酸）。多肽或蛋白质的N-末端氨基酸的α-氨基也能与FDNB反应，生成一种二硝基苯肽（DNP-肽）。由于硝基苯与氨基结合牢固，不易被水解，因此当DNP-多肽被酸水解时，所有肽键均被水解，只有N-末端氨基酸仍连在DNP上，所以产物为黄色的DNP-氨基酸和其它氨基酸的混合液。混合液中只有DNP-氨基酸溶于乙酸乙酯，所以可以用乙酸乙酯抽提并将抽提液进行色谱分析，再以标准的DNP-氨基酸作为对照鉴定出此氨基酸的种类。因此2,4-二硝基氟苯法可用于鉴定多肽或蛋白质的N-末端氨基酸。
