( Rapidly isolate...)High-Throughput Isolation of Total RNA

Rapidly isolate total RNA from 96 samples

Karen Dolter Sylvia Norman Jeff Braman
Stratagene

The StrataPrep 96 total RNA purification kit provides a convenient method for simultaneous isolation of total RNA from up to 96 cultured cell samples. Because RNA is bound to a solid support, there is no need for organic extraction and alcohol precipitation. The resulting RNA is high quality, with no detectable DNA contamination, and is ready for molecular beacon and conventional RT-PCR applications.

Isolating total RNA from many samples by traditional methods can be time-consuming and tedious. The StrataPrep method is efficient and allows processing of a wide range of sample sizes, from 10 cells to 5 x 10⁵ cells per well.

Protocol

Fig.1

An overview of the StrataPrep 96 protocol is shown in Figure 1. After cultured cells are disrupted by guanidine thiocyanate, lysates are applied to a silica-based fiber matrix within the 96-well plate. RNA is immobilized on the fiber matrix, and contaminants are removed while avoiding organic extractions and ethanol precipitation. The sample is treated with DNase directly on the fiber matrix and pure RNA is recovered by eluting with a small volume of buffer.

The standard protocol makes use of a vacuum manifold and a tabletop centrifuge. If a vacuum manifold is not available, an alternate protocol, which uses the centrifuge alone, may be used (additional materials required).

Isolation of RNA from Various Cell Lines

Table 1: Average Yields of RNA from Various Cells (g)

* Numbers in parenthesis indicate the number of isolations performed.

Species

Cell line

5 x 10⁵ cells

2 x 10⁵ cells

1 x 10⁵ cells

Mouse

NIH/3T3

11.0 (4)*

4.1 (4)

2.9 (4)

Hamster

CHO

4.7 (60)

Human

HeLa

9.2 (3)

5.5 (1)

2.1 (3)

Human

HL-60

4.0 (3)

1.9 (3)

1.1 (3)

Human

THP-1

6.4 (1)

1.6 (1)

0.8 (1)

Table 1 and Table 2 show total RNA yields from different cell numbers of a variety of cell lines. 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 samples isolated from 1 x 10⁵ to 5 x 10⁵ cells is greater than 1.9 for all samples, indicating high-quality RNA. Samples isolated from 1 x 10¹ to 1 x 10⁴ cells (Table 2) were quantitated in fluorescent assays using the RiboGreen RNA quantitation kit (Molecular Probes) since absorbance measurements cannot be taken for such small samples. These samples also reflect yields of approximately 10^-5 g of total RNA per cell.

Table 2: Average Yields of RNA from Small Numbers of Cells (ng)*

* RNA was quantitated by the RiboGreen assay using dilutions of an rRNA standard.

Numbers in parentheses indicate the number of isolations performed.

Cell line

10⁴ cells

10³ cells

10² cells

10¹ cells

HeLa

119 (6)

9.6 (6)

1.0 (6)

0.211 (2)

HL-60

53 (1)

4.8 (1)

0.53 (1)

THP-1

84 (1)

6.9 (1)

0.97 (1)

0.110 (1)

NIH/3T3

325 (4)

23.3 (4)

2.9 (4)

Representative RNAs (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 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 (C_t) 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 (C_t=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 isolation kit from Qiagen. RNA isolations were performed according to each kits instructions. The RNeasy 96 kit does not include DNase but does make available a supplemental protocol for DNase treatment; hence, we were able to carry out an RNA isolation with as well as without DNase treatment.

The StrataPrep 96 kit, with DNase treatment, generated RNA yields similar to the RNeasy 96 kit without DNase treatment (Table 3). When DNase treatment was included with the RNeasy 96 protocol, RNA yields decreased. The StrataPrep 96 kit samples have the highest RNA concentrations, which is especially important for analyzing small samples.

Table 3: Average Yields from Total RNA Purification Kits*

* RNA was isolated from NIH/3T3 cells grown in 96-well plates (approximately 3.8 x 10⁴ cells/well). The numbers are averages of three samples containing four pooled samples each. Yields were determined by spectrophotometric analysis.

The alternate protocol for the StrataPrep 96 kit was also tested (additional materials required).

Yield (g/well)

Concentration (g/ml)

A₂₆₀/A₂₈₀

StrataPrep kit

1.0 0.02

40 1.0

2.0 0.02

StrataPrep, spin only

0.9 0.07

48 2.0

2.1 0.04

RNeasy 96

1.0 0.22

16 2.3

1.9 0.05

RNeasy 96 + DNase

0.6 0.13

8 1.9

1.9 0.07

RT-PCR was performed on RNA samples isolated with both kits using primers specific for GAPDH. These primers amplify a 209-bp product from cDNA and from pseudogene sequences in genomic DNA. We performed cDNA synthesis reactions both in the presence and absence of reverse transcriptase, followed by PCR as an assay for DNA contamination. No DNA was detected in the StrataPrep 96 kit samples using either the standard or the alternate protocol, whereas both of the RNeasy 96 samples demonstrated DNA contamination, even when DNase treatment was included (Figure 5).

Fig.5

Conclusions

The StrataPrep 96 total RNA purification kit is the method of choice for isolating RNA from as many as 96 cultured cell samples. The method generates pure, intact RNA with high yields. While amplification by conventional and molecular beacon RT-PCR can be achieved for samples isolated from as few as 10 cells, the StrataPrep 96-well RNA kit is clearly superior when compared with the RNeasy kit because Stratagenes kit yields RNA at a higher concentration with no detectable DNA contamination.

Acknowledgments

The authors thank Gothami Padmabandu and Reinhold Mueller for performing molecular beacon 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. Borns, M. et al. (1999) Strategies 12: 33-36.

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

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

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