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. 2011 Feb 25;286(22):19523–19532. doi: 10.1074/jbc.M111.233916

FIGURE 2.

FIGURE 2.

End-point RT-PCR detection of HAS2-AS1 transcription, optimization of SYBR Green qRT-PCR, and absolute quantification of HAS2 and HAS2-AS1 RNAs. A, detection of HAS2-AS1 expression in PTCs by end-point RT-PCR using primers A and AR (see Fig. 1B for details of all HAS2-AS1-specific primers not used in duplex detection). B (from left to right), pairs of HAS2-AS1 qRT-PCR profiles are shown following amplification using primers SYBR-F and SYBR-R from 2 μg, 1 μg, 500 ng, and 250 ng of input RT product, respectively. Each pair comprises amplifications from RT reactions carried out using either 0.1 or 1.0 μm primer SYBR-RT. Profiles for negative control reactions with no reverse transcriptase, no RNA, and no cDNA are contained within a box that is highlighted with an asterisk. C, melting curve of HAS2-AS1 SYBR Green amplification products shown in B. The negative control reaction range is illustrated by a bracket highlighted with an asterisk. D, variation in Ct in the presence of different quantities of input RNA (see B above) and the different concentrations of HAS2-AS1-specific RT primer displayed in the key. E, absolute quantification of the copy number of HAS2 mRNA using primers aqHAS2-F and aqHAS2-R (see “Experimental Procedures” for details) and of HAS2-AS1 RNA using primers aqHAS2-AS1-F and aqHAS2-AS1-R in unstimulated cells (white bars) and in response to stimulation with IL-1β (1 ng/ml) for 3 h (black bars). Data are displayed from one of two replicate experiments, each carried out in quadruplicate, and error bars are S.D.