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Fast Isolation of Total RNA from Small Numbers of Cells

east 400 copies of GAPDH pseudogenes in the genomes of the mouse and rat and approximately 20 copies in the human genome.5,6 Figure 4 shows that DNA contamination is not detectable as demonstrated by the lack of product for reactions in which reverse transcriptase was omitted.

Fig.4

RT-PCR Analysis of RNA from Small Cell Numbers

Total RNA isolated from 105, 104, 103, 102, and 101 HeLa cells was tested as template in RT-PCR. Amplification using the human GAPDH primer set for RT-PCR with the prostar Ultra HF RT-PCR system7 was successful with all samples, including RNA isolated from only 10 cells (Figure 5). For reactions where reverse transcriptase or cDNA template was omitted, no bands were detected; therefore, the target sequence DNA did not contaminate the reactions.

Fig.5

Detecting RNA with Quantitative RT-PCR

RNA from varying numbers of HeLa and THP-1 cells was also subjected to real-time quantitative-RT-PCR analysis using a GAPDH-specific molecular beacon for detection. The molecular beacon hybridizes 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.8 The Mx4000 molecular beacon GAPDH expression analysis kit specifically detects GAPDH mRNA sequences but not genomic DNA or pseudogene sequences.9 Detecting as little as 1 pg of human total RNA or 0.01 pg of poly(A)+ RNA is possible with this kit.10 Signal was readily det
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