Fast Isolation of Total RNA from Small Numbers of Cells ( le 2 ) was quantitated in fl...)

Fast Isolation of Total RNA from Small Numbers of Cells

le 2) was quantitated in fluorescence assays using the RiboGreen RNA quantitation kit (Molecular Probes) since absorbance measurements are not sensitive enough for accurate quantitation. The quantity of RNA isolated from 10 cells is at or near the detection limit for RiboGreen (1 ng/ml as per manufacturer). Overall, RNA isolated from 10⁴ to 10¹ cells reflects yields of approximately 10^-5 g of total RNA per cell, although yield generally varies according to cell size.

Representative RNA samples isolated from 5 x 10⁵ cells (Table 1) were electrophoresed 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 ribosomal RNA bands (Figure 2).

Fig.2

RT-PCR Analysis of RNA

The high quality of total RNA isolated from 5 x 10⁵ cells was confirmed by successful RT-PCR amplification of a relatively long target. Primers specific for replication factor C (RFC) were used in RT-PCR (Figure 3) and the expected 3.1-kb band was observed. It has been reported that RFC RNA undergoes alternative processing, which leads to mRNA of other sizes.⁴ This may be the case for RNA from HeLa, HL-60, and CHO cells since additional PCR products are evident in these samples.

Fig.3

Total RNA from 5 x 10⁵ cells was also tested for the presence of contaminating DNA by RT-PCR, with and without reverse transcriptase. This assay uses GAPDH primers that amplify a 0.2-kb fragment from mRNA and genomic DNA. There are at l
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( le 2 ) was quantitated in fl...)Fast Isolation of Total RNA from Small Numbers of Cells

RT-PCR Analysis of RNA