使用方法:
1、将模板RNA和试剂在冰上解冻，使用前将每种溶液轻弹或者轻微涡旋振荡混匀，简短离心以收集残留在管壁的
液体到管底。
2、在RNasefree管中冰上配制以下反应体系:
| 组分 |
体积 |
| Total RNA/mRNA |
0.1-2 μg |
| 5xRT MasterMix |
4μl |
| 20xOligo dT &Random Primer 或特异性引物 |
1 μl |
| RNase free H20 |
补足20μl |
3、移液器轻轻吹打混匀，放入PCR仪运行如下程序:
| 快速程序 |
标准程序 |
| 37℃，15~30min |
25℃，10min |
| 85℃，5min |
55℃，30~60min |
| |
85℃，5min |
通常可选择快速程序进行反转录反应;如果模板具有复杂二级结构或高GC区域，使用标准程序。
4、得到的cDNA产物可立即用于qPCR反应，或在-20℃保存，并在半年内使用;长期存放建议分装后在-80℃保存。cDNA应避免反复冻融。
注意事项:
1、避免RNase污染。
2、为保证反转录成功建议使用高质量的RNA样品。
3、5xRT MasterMix非常粘稠，易吸附在管壁和吸头外导致损失，用前请点甩离心，并避免吸头外壁沾附损失。
4 为了您的安全和健康，请穿实验服并戴一次性手套操作。
This product is an efficient, stable, fast reverse transcription system that can remove genomic DNA contamination. 5xRTMasterMix is a one tube reverse transcription premixed Mix that contains various reagents required for reverse transcription (HRTase, RNaselnhibitor, dNTP Mixture Buffer). The reaction can be carried out by adding template RNA primers and water. The reverse transcriptase used in the kit removes RNaseH activity and has stronger thermal stability, which can withstand reactions at 55 ℃ and improve the reverse transcription of complex RNA templates. MasterMix has added a thermosensitive double stranded specific nuclease that can directly degrade and remove residual genomic DNA (gDNA) contamination in RNA samples during the reverse transcription process. Oligo dT&Random Primer single tube is provided, and if specific primers are needed, they can be directly replaced.
