Fig6G_PCR_MDCK_R2_LG401_Fig6H_PCR_P3virus_R2-R3_LG394.sgd

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.

本研究采用逆转录聚合酶链式反应（RT-PCR，Reverse Transcription Polymerase Chain Reaction）分析病毒RNA，以验证插入序列的存在。将STOML2基因的51 bp片段插入到A/Puerto Rico/8/1984 (H1N1)（简称PR8）的M基因组片段中。实验分别从三类样本中提取病毒RNA：病毒原液（G组）、感染后48小时的马-达二氏犬肾细胞（MDCK）上清液（H组），以及感染后16小时的Xrn1基因敲除A549细胞。本次分析的病毒涵盖：无插入序列的野生型PR8（WT，Wild Type）背景病毒、插入野生型STOML2序列的病毒，以及携带无法被PA-X切割的突变型STOML2序列的病毒，上述病毒均构建于野生型PR8或PR8-PA(∆X)遗传背景中。随后将提取的病毒RNA逆转录为互补脱氧核糖核酸（cDNA，complementary DNA），并使用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，以确认STOML2序列是否仍保留于病毒基因组中。