Figure 1

RT-PCR in the analyses of naturally occurring endogenous sense-antisense RNA pair expression. (a) Schematic of the cardiac MHC (MYH) gene locus and its transcription products. The upper strand transcribes the cardiac MYH7 and MYH6 sense RNA, the lower strand transcribes the antisense MYH7 RNA, which is abundant in normal control hearts [1]. (b): representative gels obtained from RT-PCR targeting sense and antisense RNA corresponding to the MYH7 gene. RT used RNase H^- enzyme under manufacturer standard conditions (see methods) in presense of specific primers (+p) or in absence of primers (-p). (c) Bar graph depicting the net signal of MYH7 sense and antisense in each group, net consisting of the difference between +p and -p RT-PCR band intensity. Note that a normal control heart in the rat is associated with abundant relatively MYH7 gene expression (MYH6 gene expression is dominant). Under the PTU condition, MYH7sense RNA expression is increased. Antisense MYH7 RNA is strongly expressed in the normal control heart, based on strong net signal. In PTU heart, the antisense MYH7 RNA is decreased to a very low level. Note the +p product is similar to -p when targeting antisense MYH7 RNA in PTU hearts. (d) Bar graph depicting relative no-primer signal (NP) to the total signal in each group as determined by real time PCR methods. (e) Net MYH7 sense and antisense RNA copy numbers in NC and PTU hearts using real time PCR. Data are means ± SE. N = 6/group. See Additional file 4 for primer information. For both sense and antisense MYH7 targets, end-point PCR (b and c) used 0.2 μl of the cDNA and was performed for 28 cycles. For real time PCR, we used 320 nl cDNA for each sample, and the signal was compared to a standard curve established with a serial dilution of a standard consisting of purified PCR product as explained in the methods. See Additional file 4 for primer information. Based on standard curve linear regression analyses, copies for each target RNA were calculated.+p: a strand specific RT primer was included; -p: RT without primer. Sense is the amplification product of the sense target obtained when the reverse primer was added to the RT reaction. Antisense is the amplification product of the antisense target obtained when the forward primer was included in the RT reaction. In all these reactions, the presence of the no primer product depended on the presence of RNA and the RT enzyme, and was not formed in RT reactions that were carried out in the absence of the reverse transcriptase enzyme.