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. Author manuscript; available in PMC: 2020 Oct 1.
Published in final edited form as: J Virol Methods. 2019 Jul 14;272:113701. doi: 10.1016/j.jviromet.2019.113701

Figure 5. Validation of a strand-specific RT-qPCR using modified tagged RT primers and PCR primer sets.

Figure 5.

(A) 10 pg of in-vitro synthesized RNAs corresponding to the genomic (G) and antigenomic (AG) segments of L, M, and S RNAs were used for cDNA synthesis using tagged RT primers specific for genomic L, M, and S segments. The corresponding cDNAs were subjected to PCR by using PCR primer sets with the ‘tag’ sequence as the forward primer and a segment-specific reverse primer that selectively amplifies genomic L (lanes 2-4), M (lanes 5-7) and S (lanes 8-10) segments. NT represents the "no template" control for RT-PCR analysis (lanes 2, 5 and 8). (B) 10 pg of in-vitro synthesized RNAs corresponding to the genomic (G), antigenomic (AG) segments of L, M, and S RNAs, and cognate mRNAs (mRNA) of L and M segments were used for cDNA synthesis by using tagged RT primers specific for antigenomic L, M, and S segments. The corresponding cDNAs were subjected to PCR using PCR primer sets with the ‘tag’ sequence as the forward primer and a segment-specific reverse primer that selectively amplifies antigenomic L (lanes 2-5), M (lanes 6-9) and S (lanes 10-12) segments. NT represents the "no template" control for RT-PCR analysis (lanes 2, 6 and 10). Lane 1 in (A) and (B) represents the DNA size marker, and the location of the markers having 100 and 200 base pairs (bp) bands are indicated by arrows. The PCR products were analyzed by agarose gel electrophoresis.