Minibest universal rna extraction kit

Total RNA was isolated using MiniBEST Universal RNA Extraction Kit (TaKaRa, Japan) and regarded as a template. The random primer Oligo (dT) was reverse-transcribed into cDNA with Primescript RT reagent kit with gDNA Eraser (perfect real time) (TaKaRa, Japan), and cDNA served as the template for qPCR. BioRad’s SYBR Green kit was used to carry out qPCR experiment. The GAPDH gene was regarded as the reference. qPCR was performed using a CFX-96 Real-Time System (Bio-Rad), at 95°C for 10 minutes, then 95°C for 10 seconds, 60°C for 15 seconds and 72°C for 20 seconds for 40 cycles. The sequences of the primers used for detecting genes are shown in Supplemental Table 1.
MiniBEST Universal RNA Extraction Kit (TaKaRa, Japan) was used to extract miRNA from the cell samples, and the reverse primers corresponding to each miRNA were used to reverse-transcribe with Primescript RT reagent kit with gDNA Eraser (TaKaRa, Japan); and the product served as the templates of qPCR. U6 was considered as the reference. qPCR was carried out according to the above method. The universal reverse primer of miRNA detected is 5ʹ-CAGTGCGTGTCGTGGAGT-3ʹ. The reverse primers and forward primers of miRNA measured are displayed in Supplemental Table 2. All experiments were repeated three times in each group; 2^−ΔΔCt was used in the quantification of mRNA and miRNA.

To evaluate the differentiation status of AOs cultured in PD medium versus those in AO medium, the organoids were harvested at the indicated times, and RNA was extracted using MiniBEST Universal RNA Extraction Kit (TaKaRa). To quantify virus replication, the organoids and supernatant samples were lysed for RNA extraction using the MiniBEST Universal RNA Extraction Kit and MiniBEST Viral RNA/DNA Extraction Kit (TaKaRa), respectively. cDNA was synthesized with the Transcriptor First Strand cDNA Synthesis Kit (Roche) with Oligo-dT primer. qPCR was performed with LightCycler 480 SYBR Green I Master Mix (Roche) using gene-specific primers (SI Appendix, Table S2) to detect cellular gene-expression level and viral gene copy number. The mRNA expression levels of cellular genes were normalized with that of GAPDH. Viral gene copy number was determined by absolute quantification using a plasmid expressing a conserved region of the IAV M gene.