Figure 4

One Step RT-PCR systems and specificity for the analyses of sense-antisense RNA pairs. (a) Comparisons of four commercially available one step RT-PCR kits regarding their performance in analyses of sense and antisense MYH7 RNA expression in NC and PTU heart total RNA. -P is when the RT step was carried out in absence of primers. +R is when the RT was carried out in presence of reverse primer that is targeting the sense RNA. +F is when the RT was carried out in presence of forward primer that is targeting the antisense RNA. (b). One Step RT-PCR using the Qiagen kit. Reactions used human brain RNA and PCR targeting either sense or antisense RNA of specific genes shown to be expressed in sense/antisense pairs (SA22, SA24) or in a single form (NC3, NC10). These primers were used previously by Chen et al., [9]. As a further test of specificity, the RT reactions were carried out in absence of primers (-P) or in presence of a non specific primer (+N), complementary to human MYH4 mRNA, MYH 4 is not expressed in the brain and is not related to any of the studied genes. (c) One step RT-PCR (Qiagen kit) used to detect small amount of antisense (AS) MYH 7 RNA in mixed total RNA preparations containing either 100% sense (S) MYH7 RNA, (100:0), or 99:1 and 90:10 of S:AS ratios. Sense MYH7 is derived from soleus muscle total RNA, whereas antisense MYH7 is derived from total RNA in T3-treated rat hearts. Reactions contained 100 ng of total RNA of the specified S:AS composition. RT reaction included either no primers (-P), a forward primer (+F) to target the antisense, and a non-specific primer (+N). For all these reactions in a, b, and c, 100 ng total RNA was used per reaction, and the RT was carried out at 50°C for 30 minutes followed by 10 minutes at 95°C. The PCR was carried out for 30 cycles (a, b, and c) or 35 cycles (c). See Additional file 4 for primer information.