RT-PCR analyses

Fl. (8). RT-PCR analyses of TNF-a and/or croc in-treated PC-12 cell lysates for the mRNA expression of... 

Flg.(9). RT-PCR analyses of TNF-a and/or crocin-treated PC-12 cell lysates for the mRNA expression of pro-apoptotic proteins, Bax, Bcl-Xs and LICE. The experimental procedures were those described in Materials and methods. The bands in agarose gel were visualized and photographed under UV radiation. The photographs were scanned and quantified by using an NIH Imager. The expression of GAPDH mRNA was used as a control. Each bar represents the mean S.D. of three independent experiments. P<0.01 P<0.001, compared to the control. P<0.05 M P<0.01 P<0.001, compared to TNF-aalone. 

Northern blot and RT-PCR analyses showed that the A9 desaturase-encoding RNA was present in T. ni adult and larval fat body, as well as in pheromone-gland and muscle tissue. Interestingly, no A9 desaturase-encoding cDNA was... 

More detailed RT-PCR analyses of mRNA from a variety of tissues have revealed additional structural complexity and identification of a large number of alternatively spliced subunit transcripts that differ in variable domain nucleic acid sequence (Tombes et al. 2003). These subunit variants are expressed in a tissue-specific manner. For example, the 6i subunit mRNA is expressed predominantly in brain, whereas S2 and 63 variants are expressed in rat aortic smooth muscle,... 

Figure 5.3. TNF R1 screen. Results from a screen to identify the optimal antisense inhibitor to tumor necrosis factor a receptor 1 (TNFa-Rl). 2 -Methoxyethyl chimeric antisense inhibitors were synthesized to 80 sites in the target RNA and their effects on TNFa-Rl, RNA were evaluated after incubation with TNFa-Rl-expressing cells. The results are expressed as percentage control RNA determined by RT-PCR analyses. The effects of the antisense inhibitors are displayed schematically from 5 UTR to 3 UTR right to left on the figure. The antisense inhibitors that result in the maximum reduction in target RNA (smallest bars) are then evaluated with careful dose-response curves and transcriptional arrays along with control oligonucleotidesto ensure the effects are specific.

Mannhalter C, Koizar D, and Mitterbauer G (2000) Evaluation of RNA isolation methods and reference genes for RT-PCR analyses of rare target RNA. Clinical Chemistry and Laboratory Medicine 38 171-177. 

For sake of simplicity this procedure will descnbe RT/PCR (see Fig. 3B for a schematic) for human RARa isoforms. Figure 5 compares the results of such analysis to those obtained with Northern blot using the same panel of different cell lines. Table 1 lists all RT and PCR primers required to carry out a comparable RT/PCR analyses on all major murine RAR isoforms as well as mouse RXRa, P and y. To avoid confounding results due to amplification of RAR/RXR sequences that may be present in small amounts of contaminating-genomic DNA, the 5 and 3 oligonucleotide primers for PCR amplification were derived from regions encoded in separate exons. 

Figure 4. Dot-blot hybridization and RT-PCR analysis of peg operon in strain 103, (A) Dot-blot hybridization was performed using gene-specific probes generated by PCR, The sizes of the probes were identical to products obtained by RT-PCR analyses (B), (B) RT-PCR analyses of each gene and (C) RT-PCR analyses of each intergenic region were performed.

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