对差异m6a基因/转录本进行qpcr验证是表观转录研究必不可少的步骤。康成生物提供与merip-seq相对应的merip-pcr服务,保证最优的实验流程与最佳的技术方案。
图一. merip-pcr实验流程图。
康成丨数谱生物服务特色:
●优化的实验流程
●特异性强的m6a抗体
●通过严格测试的引物
merip-pcr既可对完整的m6a转录本(intact)进行检测,也可对该转录本的m6a修饰区域(m6a-deposited fragments)进行检测。
(zhang et al., 2017, cancer cell 31, 1–16)
normalize each merip rna fractions’ ct value to the input rna fraction ct value for the same qpcr assay (δct) to account for chromatin sample preparation differences. calculate the % input for each merip fraction:
%input=2(ct input-ct merip)×fd×100% here, fd is input dilution factor.
for example, if one- thirtieth of input mrna and one-fifth of merip mrna were used for qpcr assays, fd =5/30 =1/6
fold enrichment=2[ pos(ct input-ct merip)-neg (ct input-ct merip) ]
sample |
input-ip |
input-neg |
ip/neg |
||||
input(ct) |
ip(ct) |
% |
input(ct) |
neg(ct) |
% |
||
2fvector c1 |
27.515 |
32.061 |
0.71 |
27.515 |
na |
0.01 |
61.35 |
2f4e c3 |
27.287 |
32.885 |
0.34 |
27.287 |
na |
0.01 |
34.65 |
2fvector c5 |
26.652 |
25.811 |
29.85 |
26.652 |
na |
0.01 |
4669.09 |
2f4e c6 |
26.018 |
25.145 |
30.52 |
26.018 |
na |
0.00 |
7408.39 |
2f4e c3/2fvector c1 |
|
0.48 |
|
0.85 |
|
||
2f4e c6/2fvector c5 |
|
1.02 |
|
0.64 |
|