Testing different RT enzyme properties and conditions. (a) In a two-step RT-PCR system, 2 μg total RNA from NC and PTU treated hearts were reverse transcribed in 20 μl reactions in absence (-p) or presence (+p) of RT primers. The RT primer targeted the MYH7 sense RNA, and the PCR primer set amplified a 284 bp product corresponding to the 3' end of the MYH7 gene. PCR used 1 μl cDNA and was carried out for either 28 or 30 cycles. Shown are results from using two different RT enzymes that differed by their RNase H properties. RNase H- and RNase H+. For each enzyme, the RT reactions were carried out under two different temperatures: 44°C or 50°C for 30 minutes/ea. (b) RT-PCR targeting the antisense MYH7 RNA in total RNA mixes of known proportions of sense and antisense RNA. RNA template contained either only sense MYH7 RNA, or a mix of sense and antisense MYH7 RNA corresponding to 99 to1 or 90 to 10 sense to antisense ratios (S:AS). Soleus total RNA was used as a source of the sense MYH7 RNA in absence of antisense. Whereas, T3-treated heart total RNA was used as a source of the antisense MYH7 RNA without co-expression of the sense. Mixes of soleus and T3 treated heart RNA were used to achieve the noted S:AS amounts in 2 μg of total RNA per 20 μl reactions. Reverse transcriptions were carried out in absence of RT primers (-P), in presence of the forward primer (+F) targeting the antisense, and in presence of a non specific primer corresponding to the 3' untranslated region of the human MYH4 mRNA sequence (+N). RT reactions used RNase H- RT (Invitrogen), performed at 44°C or at 50°C for 30 min. PCR was carried out on 1 μcDNA for 28 cycles targeting the 3' end of the MYH7 gene. See Additional file 4 for primers information.