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High-Throughput Isolation of Total RNA

in formaldehyde-agarose gels to check the integrity of the RNA. All of the samples are intact according to this analysis, as observed by the distinct rRNA bands (Figure 2).

Fig.2

RT-PCR Analysis of RNA from HeLa Cells

Total RNA isolated from various numbers of HeLa cells was tested as a template in RT-PCR. Amplification using the human GAPDH primer set for RT-PCR with the prostar Ultra HF RT-PCR system4 was successful with all samples, including RNA isolated from only 10 cells (Figure 3). The absence of bands for negative control reactions shows that the PCR reactions are not contaminated with the target sequence.

Fig.3

Detecting Low-Abundance RNA Using Molecular Beacon RT-PCR

RNA from varying numbers of HeLa cells was also subjected to molecular beacon RT-PCR analysis5 using primers and a beacon specific for the low-abundance target HPRT (Figure 4). The beacon hybridizes quantitatively to the target sequence and emits fluorescence in this conformation. The threshold cycle number (Ct) is inversely proportional to the concentration of target sequence in the reaction.6 This low-abundance target was readily detected from an RNA sample isolated from as few as 10 cells (Ct=35).

Fig.4

Comparison of 96-Well Total RNA Isolation Kits

DNA contamination of RNA samples can interfere with RT-PCR since DNA may serve as a template during PCR. Therefore, it is imperative to remove DNA during RNA isolation. Total RNA was isolated from NIH/3T3 cells grown in 96-well plates using the StrataPrep 96 total RNA purification kit and the RNeasy 96 total RNA
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