Takara Bio"s PrimerArray Series offers primer sets for real-time RT-PCR (qPCR) that can be used for gene expression analysis of biological pathways. Each PrimerArray Series primer set contains 96 total primers that allow for the simultaneous analysis of 88 biological pathway-related genes and 8 housekeeping genes. The performance of each primer set for real-time RT-PCR (qPCR) has been validated. Primer set information can be downloaded by opening the product table and clicking on the Resources tab for your product of interest.
Takara Bio"s PrimerArray Series offers primer sets for real-time RT-PCR (qPCR) that can be used for gene expression analysis of biological pathways. Each PrimerArray Series primer set contains 96 total primers that allow for the simultaneous analysis of 88 biological pathway-related genes and 8 housekeeping genes. The performance of each primer set for real-time RT-PCR (qPCR) has been validated.
The PrimerArray Analysis Tool* (download) is capable of analyzing data obtained using PrimerArray Series products. This analysis tool is linked to the KEGG (Kyoto Encyclopedia of Genes and Genomes) database, allowing results to be visualized on a pathway map. Primer set information and a manual on how to use the tool can be downloaded by opening the product table and clicking on the Resources tab for your product of interest.
* The PrimerArray Analysis Tool uses a macro. Please select "enable macro" each time to use the file.
Available PrimerArray Primer Sets
Human
- PH001 - Cytokine-cytokine receptor interaction
- PH002 - Cell cycle
- PH003 - Cell adhesion molecules (CAMs)
- PH004 - Jak-STAT signaling pathway
- PH005 - Natural killer cell mediated cytotoxicity
- PH006 - Axon guidance
- PH007 - Focal adhesion
- PH009 - TGF-beta signaling pathway
- PH010 - Wnt signaling pathway
Mouse
- PN001 - Cytokine-cytokine receptor interaction
- PN002 - Cell cycle
- PN003 - Cell adhesion molecules
- PN004 - Jak-STAT signaling pathway
- PN005 - Natural killer cell mediated cytotoxicity
- PN006 - Axon guidance
- PN007 - Focal adhesion
- PN008 - T-cell receptor signaling pathway
- PN009 - TGF-beta signaling pathway
- PN010 - Wnt signaling pathway
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看引物能不能特异性扩增,你可以检查一下扩增后的熔解曲线,一般单一的峰是特异性引物,如果有双峰那就是扩出来了杂带
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
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。
在分子生物学中,探针是根据碱基互补的原理,用来与特定的DNA片段做杂交以对特定的DNA片段进行检测,如Southern杂交
引物是用来扩增DNA序列的,因为核苷酸必须要连接在3-OH上才能够合成延伸,引物就是用来提供3-OH的。
只要是符合要求的DNA片段都可以用来做探针和引物,引物是用来扩增DNA序列,探针用来与特定的DNA片段做分子杂交的
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。

