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jghu9200

木虫 (小有名气)

[求助] Reviewer提的两个问题,求如何回答,如何做?万分感谢

Reviewer提的两个问题,本人对这个问题有点拿不准,求热心战友帮忙,如何回答?,如何做?万分感谢

Reviewer #2: The authors did address most concerns of the reviewer. However, there is one technical issue which has to be clarified.

1. In the MM section, the authors state that "To ensure that the samples were free of genomic DNA, RNA samples were subjected to a reverse transcription in the absence of the reverse transcriptase. Our results didn't find any genomic DNA contamination." The authors are correct to assume that if there is any contamination with genomic DNA, then there should be amplification in the subsequent PCR. HOWEVER, in case the primer is an exon-intron spanning oligonucleotide and the amplified intron is too long, than this genomic contamination band will not be visible in a 1% agarose gel. I strongly suggest performing another control experiment with contaminated RNA to ensure that the assay performed is sufficient to detect genomic DNA contamination.

3. Which strategy was followed to evaluate real-time PCR experiments?
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jghu9200

木虫 (小有名气)

引用回帖:
2楼: Originally posted by fuyuandj86 at 2013-09-27 11:21:50
第一个说得很明白吧?让你补试验你就老实补吧.虽然似乎有点挑剔
我理解就是让你加个阳性对照,把RNA里面故意加入一点genomic DNA.然后用你的引物应该可以在胶上看到污染.

谢谢
4楼2013-09-28 21:27:50
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jghu9200

木虫 (小有名气)

引用回帖:
3楼: Originally posted by niil at 2013-09-27 14:00:30
1. 审稿者认为你的实验的对照有问题。你的对照是不加反转录酶,如果有条带的话,就是DNA污染,但是,由于DNA含有内含子,如果内含子长度过长而且你的引物是跨内含子设计的,那么你依旧不能扩出条带了,但是这种情况 ...

非常感谢
5楼2013-09-28 21:28:38
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