Response to: Elevated L1 expression in ataxia telangiectasia likely explained by an RNA-seq batch effect.
Response to: Elevated L1 expression in ataxia telangiectasia likely explained by an RNA-seq batch effect.
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反应：共济失调毛细血管扩张症中 L1 表达升高可能是通过 RNA-seq 批次效应来解释的。
DOI：
10.1016/j.neuron.2023.02.006
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发表时间：
2023
期刊：
影响因子：
16.2
通讯作者：
Glaz
中科院分区：
文献类型：
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作者：
Takahashi,Takehiro;Stoiljkovic,Milan;Song,Eric;Gao,Xiao-Bing;Yasumoto,Yuki;Kudo,Eriko;Carvalho,Fernando;Kong,Yong;Park,Annsea;Shanabrough,Marya;Szigeti-Buck,Klara;Liu,Zhong-Wu;Kristant,Ashley;Zhang,Yalan;Sulkowski,Parker;Glaz
Faulkner博士在他的信中认为，共济失调毛细血管扩张症（AT）患者的小脑L1 HS并没有显著增加。它指出AT中的内含子读段增加，这与L1读段相关;因此，增加的L1读段反映了更高的内含子读段。这封信认为，人为因素，如RNA质量可能是报告的差异的原因，我们对人类样本的发现应该从论文中省略。我们感谢福克纳博士对我们研究的兴趣，我们已经进行了额外的实验来解决这些问题。我们的新结果支持和加强了我们的研究的原始结论，对于L1亚家族的归一化计数之和的比较，at检验可能不是最好的统计方法。然而，AT中存在明显的L1 HS增加趋势，这与整个队列中qPCR的显著差异一致。1这封信批评了L1 HS qPCR，我们使用了针对Orf 1/2的引物，而不是50个UTR，并引起了对基因组DNA（gDNA）污染的担忧。我们使用靶向50个UTR 2的引物进行qPCR，并再次发现AT中的L1 HS显著升高（图S1 A）。我们包括其中未将逆转录酶（RT）添加到从RNA合成cDNA步骤中的样品（无RT对照）。无RT样品的Ct值通常比有RT对应cDNA样品高10个循环，表明有RT样品中的gDNA污染仅占qPCR值的2 × 10
In his letter, Dr. Faulkner argues that L1HS is not significantly increased in ataxia telangiectasia (AT) patients’ cerebella. It notes that there are increased intronic reads in AT, which correlate with L1 reads; thus, increased L1 reads are the reflection of higher intronic reads. The letter contends that artifactual factors such as RNA qualities might be the cause of reported differences, and our findings with human samples should be omitted from the paper. We thank Dr. Faulkner for his interest in our study, and we have performed additional experiments to address these issues. Our new results support and strengthen the original conclusions of our study.For the comparison of the sum of normalized counts of L1 subfamilies, at test may not have been the best statistical approach. Nevertheless, there is a clear trend of increased L1HS in AT, which is in line with significant differences with qPCR for entire cohorts. 1 The letter criticizes that, for L1HS qPCR, we used primers targeting Orf1/2, but not 50 UTR, and raises concern for genomic DNA (gDNA) contamination. We performed qPCR using primers targeting 50 UTR 2 and again found a significant L1HS elevation in AT (Figure S1A). We included samples in which reverse transcriptase (RT) was not added to the cDNA synthesis step from RNA (no-RT control). No-RT samples generally had Ct values higher by 10 cycles compared to with-RT counterpart cDNA samples, suggesting that gDNA contamination in with-RT samples constituted only 2 À10 of the qPCR values