Fig6G-6I_PCR_MDCK_A549_R3_LG403-404_annotated.tif

RT-PCR analysis of viral RNA to verify presence of inserted sequences. A 51 bp segment of the STOML2 gene was inserted in the M segment of A/Puerto Rico/8/1984 (H1N1) ("PR8"). Viral RNA was extracted from viral stocks (G), supernatants from MDCK cells 48 hours post infection (H), or Xrn1 knock-out A549 cells 16 hours post-infection. Viruses analyzes included  viruses harboring no insert, inserted WT STOML2 or mutant STOML2 sequences that is not cut by PA-X, either in the WT PR8 or PR8-PA(∆X) background. The RNA was then reverse-transcribed to cDNA and PCR amplified using primers located on either side of the STOML2 sequence to visualize on an agarose gel whether the STOML2 sequence was retained.

为验证插入序列的存在，本研究采用逆转录聚合酶链式反应（RT-PCR）对病毒RNA开展分析。将STOML2基因（STOML2 gene）的51 bp片段插入至A/波多黎各/8/1984（H1N1）（以下简称PR8）的M节段中。
分别从以下三类样本中提取病毒RNA：病毒原液（G组）、感染后48小时的MDCK细胞上清液（H组），以及感染后16小时的Xrn1基因敲除A549细胞。
本次分析涵盖的病毒包括：无插入序列的野生型PR8、携带野生型STOML2插入序列的病毒，以及携带无法被PA-X蛋白（PA-X）切割的突变型STOML2插入序列的病毒；上述病毒均分别在野生型PR8或PR8-PA(ΔX)的遗传背景下构建。
随后将提取的RNA逆转录为互补DNA（cDNA），并利用STOML2序列两侧的引物进行PCR扩增，通过琼脂糖凝胶电泳可视化检测STOML2序列是否仍保留于病毒基因组中。