( Extract total RNA from as few as ...)Fast Isolation of Total RNA from Small Numbers of Cells

Extract total RNA from as few as 1 cell

Karen Dolter Jeff Braman
Stratagene

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.

Easy Protocol

Fig.1

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 necessary buffers and components for total RNA purification including lyophilized DNase I for convenient room temperature storage.

Isolating RNA from Different Sources

Average yields of RNA from various cells

Cell line

5 x 10⁵ cells

1 x 10⁵ cells

Yield (g)

A₂₆₀/A₂₈₀

Yield (g)

A₂₆₀/A₂₈₀

HeLa

10.1

2.0

2.1

2.0

HL-60

2.8

2.1

0.5

2.1

THP-1

4.1

2.0

0.7

2.1

NIH/3T3

7.3

2.0

1.5

2.1

CHO

5.7

2.0

1.2

2.1

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.

Average Yields of RNA* from Smaller Numbers of Cells

Cell line

10⁴ cells

10³ cells

10² cells

10¹ cells

Yields (ng)

HeLa

243

28

ND**

0.4

HL-60

86

8.2

0.8

0.2

THP-1

88

10.3

0.8

0.3

NIH/3T3

176

17.0

1.1

1.0

CHO

154

12.5

1.4

0.5

* RNA was quantitated by RiboGreen assay. Isolations were performed in duplicate.
** Not done

Total RNA from smaller samples (Table 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 least 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 (C_t) 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 detected from one tenth of the RNA sample isolated from a one-cell equivalent, with C_t values of 35 (HeLa) and 36 (THP-1) (Figure 6). As expected, the C_t value increases with a decrease in cell number.

Fig.6

Conclusions

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.

Acknowledgments

The authors thank Robert Saiz and Reinhold Mueller for performing molecular beacon RT-PCR and Sylvia Norman for assisting with conventional RT-PCR.

REFERENCES

1. Sambrook, J., et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

2. Steege, M. and Cayouette, M. (1996) Strategies 9: 53-56.

3. Cayouette, M. and Hansen, C. (1996) Strategies 9: 56-57.

4. Luckow, B., et al. (1994) Mol. Cell. Biol. 14: 1626-1634.

5. Sabrouty, S. R.-E., et al. (1989) J. Mol. Evol. 29: 212-222.

6. Piechaczyk, M., et al. (1984) Nature 312: 469-471.

7. Borns, M., et al. (1999) Strategies 12: 33-36.

8. Cayouette, M., et al. (1999) Strategies 12: 85-88.

9. Cayouette, M., et al. (1999) Strategies 12: 89-92.

10. Padmabandu, G. and Mueller, R. (1999) Strategies 12: 94-97.

'"/>

Source:


Page: All 1 2 3 4 5 6
Related biology technology :

1. Fast and Easy Isolation of PCR-Ready Genomic DNA from Whole Blood
2. High-Throughput Isolation of Total RNA
3. Simple Isolation of RNA from Tissue and Cultured Cells
4. Comparison of Growth Techniques and Media for the Purpose of Plasmid Isolation from E. coli Using the Eppendorf Perfectprep Plasmid Mini Kit
5. Isolation of Microorganisms
6. Isolation of Genomic DNA from Saliva Using the Perfect gDNA Blood Mini Kit
7. Mouse Tail Genomic DNA Isolation Protocol(1)
8. Genomic DNA Isolation Protocol(1,2,3)
9. Basic Plasmid DNA Isolation Protocol
10. Protocol for RNA Isolation Using TRIzol Reagent with Phase Lock Gel-Heavy
11. RNA-free Plasmid DNA Isolation Protocol