Extract total RNA from as few as 1 cell
Karen Dolter Jeff Braman
The StrataPrep total RNA microprep kit provides a convenient method for isolating total RNA from small samples of cells. For the purification, RNA is bound to a solid support, which eliminates the need for organic extraction and alcohol precipitation. The resulting RNA is high quality, with no detectable DNA contamination and is suitable for subsequent quantitative or conventional RT-PCR.
Stratagenes recently released StrataPrep total RNA miniprep kit is designed to isolate total RNA from cells ranging from 105 to 107 cells. However, when you need total RNA from a smaller sample, the StrataPrep total RNA microprep kit is ideal. In the past, isolating total RNA from very small samples by traditional methods was difficult and inefficient because yields from small samples tend to be low due to losses during phenol-chloroform extraction and/or ethanol precipitation. Additionally, organic solvents are hazardous. Now, with Stratagenes StrataPrep total RNA microprep kit, total RNA can be extracted from as few as 10 cells to as many as 5 x 105 cells.
As shown in the Figure 1 overview, cultured cells are disrupted by guanidine thiocyanate, then bound to a silica-based fiber matrix within a spin cup. RNA is immobilized on the fiber matrix, which allows contaminants to be removed while avoiding organic extractions and ethanol precipitation. DNA is removed with a single DNase I incubation step directly on the matrix, the matrix is washed, and pure RNA is eluted with a small volume of buffer. The recovered RNA is then ready to use. The StrataPrep total RNA microprep kit provides all nece ssary buffers and components for total RNA purification including lyophilized DNase I for convenient room temperature storage.
5 x 105 cells
1 x 105 cells
Table 1 and Table 2 show total RNA yields from varying cell numbers of different cell types. A typical mammalian cell contains approximately 10-5 g of total RNA.1 The yields for all cell lines are close to or higher than this number, indicating high yields. The A260/A280 ratios measured for all samples isolated from 5 x 105 or 1 x 105 cells were 2.0 to 2.1, indicating high-quality RNA.
* RNA was quantitated by RiboGreen assay.
Isolations were performed in duplicate.
** Not done
Total RNA from smaller samples (Tab 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).
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.
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 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.
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.
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 ected from one tenth of the RNA sample isolated from a one-cell equivalent, with Ct values of 35 (HeLa) and 36 (THP-1) (Figure 6). As expected, the Ct value increases with a decrease in cell number.
The StrataPrep total RNA microprep kit is the method of choice for isolating RNA from small cell samples. The protocol and supplied reagents provide a method to purify pure, intact RNA with high yields. Amplification by conventional RT-PCR is feasible from as few as 1 cell and by quantitative RT-PCR from only one cell.
The authors thank Robert Saiz and Reinhold Mueller for performing molecular beacon RT-PCR and Sylvia Norman for assisting with conventional RT-PCR.
Sambrook, J., et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Steege, M. and Cayouette, M. (1996) Strategies 9: 53-56.
Cayouette, M. and Hansen, C. (1996) Strategies 9: 56-57.
Luckow, B., et al. (1994) Mol. Cell. Biol. 14: 1626-1634.
Sabrouty, S. R.-E., et al. (1989) J. Mol. Evol. 29: 212-222.
Piechaczyk, M., et al. (1984) Nature 312: 469-471.
Borns, M., et al. (1999) Strategies 12: 33-36.
Cayouette, M., et al. (1999) Strategies 12: 85-88.
Cayouette, M., et al. (1999) Strategies 12: 89-92.
Padmabandu, G. and Mueller, R. (1999) Strategies 12: 94-97.