英语翻译2.7Real time PCR primers were designed using the PrimerS

英语翻译
2.7Real time PCR primers were designed using the PrimerSelect program of the
DNASTAR software.The primer pairs for each gene were:Bm14-3-3ζ Forward
CGGCCGCGATGAAGGAA,Reverse GCCCAAAACGTCGTAGCAGATT;
Bm14-3-3ε Forward CGGACTCGCGCTCAACTTCT,Reverse CGCCGGCTCCTTCTGCT;
18S rRNA Forward CGATCCGCCGACGTTACTACA,Reverse
GTCCGGGCCTGGTGAGATTT.SuperScript™ III Platinum® SYBR®
Green One-Step qRT-PCR Kit with ROX (Invitrogen) was used for real time
PCR.15 μl reaction mixtures contained 7.5 μl 2×SYBR-Green Reaction mixed
with Rox,0.3 μl Superscript™III RT/platinum® Taq mix,1.2 μl 1 μM forward
and reverse primers,respectively,1 μl total RNA and 3.8 μl DEPC-water.Realtime
PCR was performed by ABI Prism 7300 Sequence Detection System
(Applied Biosystems) under the following PCR conditions:an initial cycle at
50 °C for 3 min,one cycle at 95 °C for 5 min,followed by 40 cycles of 95 °C for
15 s,59 °C for 15 s and 72 °C for 30 s.In a 96-well plate,each reaction was
performed in triplicate along with the endogenous 18S rRNA control gene.At the
end of the real-time PCR cycles,dissociation curve was performed to check for
the presence of non-specific dsDNA SYBR Green hybrids,such as primerdimers.
Data analysis was performed using ABI Prism 7300 SDS Software
V1.3.1 (Applied Biosystems,USA).Expression levels of the target genes were
normalized against the expression level of the 18S rRNA gene.The relative
expression level was calculated using 2−ΔΔCT where ΔΔCT=(CT,target gene
−CT,18S rRNA) different stages or tissues−(CT,target gene−CT,18S rRNA)
maximum.

2.7实时聚合酶链反应 被使用于DNASTAR软件的优先选择程序.每个基因的引子对为：
Bm14-3-3ζ Forward
CGGCCGCGATGAAGGAA, Reverse GCCCAAAACGTCGTAGCAGATT;
Bm14-3-3ε Forward CGGACTCGCGCTCAACTTCT, Reverse CGCCGGCTCCTTCTGCT;
18S rRNA Forward CGATCCGCCGACGTTACTACA, Reverse
GTCCGGGCCTGGTGAGATTT.SuperScript™ III Platinum® SYBR®
带有 ROX （常用生化试剂和分子生物学产品）的绿色一步式 qRT-PCR Kit (Invitrogen) 被用于实时聚合酶链反应.15 μl 反应混合物包含带有 ROX 的7.5 μl 2×SYBR-Green 反应混合物,0.3 μl 的混合Superscript™III RT/platinum® Taq ,1 μl total RNA and 3.8 μl DEPC-water.在聚合酶链反应条件下通过ABI 三棱镜 7300序列监测系统（应用生物系统）进行聚合酶链反应：原始周期为50 °C , 3 min,一个5min的95 °C周期,接着是40个15秒的95 °C ,15秒的59 °C,30秒的 72 °C .在96孔板中,每个反应在三棱镜下运行,伴以内在的18S rRNA控制基因.在实时聚合酶链反应周期的最后,运行溶解曲线来检测有无像高等二聚物一样的特别dsDNA SYBR 绿色胚胎的存在.
利用ABI Prism 7300 SDS 软件V1.3..1（美国应用生物系统）运行数据分析.目标基因的基因表现相比18S r核糖酸基因的基因表现恢复正常.相对的基因表现运用 2−ΔΔCT 计算.
ΔΔCT=(CT, target gene
−CT, 18S rRNA) 组织不同阶段−(CT, 目标基因−CT, 18S rRNA)
最大值.