RT-PCR products

Figure 7.3 UV versus LIF detection of the CE separation of a 53 base pair RT-PCR product from the RNA of the polio virus vaccine, Sabin 3. An Hae Ill-digested d>X174 DNA marker was coinjected with the PCR product for size determination—note the 72 bp fragment. The same Sabin 3 concentration was used for each analysis, whereas the marker total DNA concentration varied from 200 mg/mL for UV analysis, to 20 mg/mL for LIF analysis. Note the unambiguous pattern observed with LIF for the Sabin 3 fragment compared to UV detection of the same fragment. Full scale UV detection, 0.005 absorbance unit (AU) LIF detection, 10 relative fluorescence units (RFU). [Reproduced with permission from Schwartz et al., J Capillary Electrophor 1 36 (1994). Copyright ISC Technical Publications, Inc.]...

Figure 7.9 CE separation and quantitation of the RT-PCR product (53 bp) from Sabin 3 strain of the polio virus vaccine. Discrete volumes of the template RNA were reverse transcribed and the complementary DNA PCR amplified. The resulting products were analyzed by CE-LIF using a replaceable gel matrix. With increasing amounts of RNA template, peak height and area also increased up to point (> 2.0 /nL template), whereupon the reaction plateaued.

Rossomando EF, White L, Ulfelder KJ (1994) Capillary electrophoresis Separation and quantitation of RT-PCR products from polio virus. J Chromatogr B 656 159-168. 

The experiments in this section describe the procedures to obtain a high-titer recombinant virus stock either through recombination of RT-PCR product with RT-deleted proviral DNA or PRO-PCR product with PRO deleted proviral DNA. The susceptibility testing is outlined in Subheading 4. The following steps are described (Fig. 1) ... 

The system was demonstrated by performing an amplification of the p-globin gene from human genomic DNA isolated from whole blood. The RT-PCR products were analyzed off-chip using agarose gel (2%) electrophoresis and ethidium bromide staining. 

Figure 9 RT-PCR analysis of cytokine mRNA expression in the spleens of DBA/2 mice immunized with casein/CKA. Total RNA was extracted from pooled spleens of S DHA/2 mice or CVE-administercd mice 3 weeks after immunized using RNAzol and reverse-transcribed, and the cDNA concentration was adjusted by co-amplification of two fold serially diluted cDNA and constant amounts of control plasmid pMCQ with [5-actin specific primers. Cytokine expression patterns of IL-12, IFNy, IL-6, and 11.-10 were determined by PCR using murine cytokine-specific primers. The PCR products were separated in 2 % agarose gel and visualized by ethidium bromide staining. Relative quantification of the amounts of the RT-PCR products was performed using a Computing Densitometer and NIH Image software,...

Fig. 1. Induction of MHC class II antigens on PMN in culture. PMN were cultivated with AIM-V, 2.5% heat-inactivated autologous serum with or without IFN-y (100 U/ml) plus GM-CSF (50 U/ml). a Surface expression of MHC class II was measured after 24 and 48 h. An antibody to CD66b (former CD67) was used to identify the PMN population isotype controls are on the left the middle panel shows PMN without cytokines, b RT-PCR products generated using MHC class Il-specific primers and PMN-derived RNA are shown. PMN had been cultured for 24 h with AIM-V/NHS (1), +IFN-7 (100 U/ml) (2), and GM-CSF (50 U/ml) (3). The left panel shows the respective RT-PCR products for P-actin. c By coirfocal laser microscopy and cytofluorometry it was seen that only a subpopulation of PMN acquired MHC class II antigens, d Induction of MHC class II antigens on PMN in whole blood. Whole blood (heparinized peripheral blood of healthy donors) was cultivated with IFN-7 (100 U/1 ml blood) for 48h then upregulation of MHC class II on PMN was measured by cytofluorometry and compared to that obtained with PMN isolated from the same donor. D1-D5 designate five individual donors.

Partial RT-PCR products are used as Northern blotting probes. Probes are hybridized to nylon membranes that had been blotted with 40 (tg of human brain total RNA or 10 (tg of human hippocampal poly A+ RNA (Clontech, Palo Alto, CA). 

Human brain RNAs from different region were used as quantitative RT-PCR templates. The ratio between RT-PCR products corresponded to that of iso-forms. 

A popular technique used to observe gene expression is reverse transcriptase-polymerase chain reaction (RT-PCR). RT-PCR works by reverse transcribing mRNA to a complementary DNA sequence that corresponds to the gene of interest. Several years ago, Zabzdyr and Lillard showed that CE could be used to separate and detect RT-PCR products corresponding to P-actin in individual... 

FIGURE 14.7 Electropherogram of multiplex RT-PCR products correspoudiug to P-actin and the a estrogen receptor from single human breast carcinoma cells. (From Zabzdyr, J.L. and LiUard, S.J., Electrophoresis, 26, 137, 2005. With permission.)... 

Figure 7 RT-PCR products electrophoresed on an agarose gel. From left to right, each of the three pairs of lanes represents, respectively, mRNA levels for (1) prepro-tachyldnin (PPT) (2) the 3 nicotinic receptor suhunit, both in the PG and (3) TH in the carotid body. Transcript (mRNA) levels for chronic hypoxia (CH 9-14 days at 380torr) versus normoxia (N) are represented by the left versus right lanes, respectively, for each pair. Right lane shows DNA molecular-weight markers and indicated base pair sizes.

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