( ...)Easy preparation of total RNA from cultured cells with TRIzol and ,,, Eppendorf Phase Lock Gel

Christian Bauer, Oliver Klotzsche, and Frank Apostel
Christian Bauer, Oliver Klotzsche, CCS Cell Culture Service GmbH, Falkenried 88, D-20251 Hamburg, Germany,
Frank Apostel, Eppendorf AG, Germany Introduction

The classic RNA isolation procedures are based on a mono-phasic solution of phenol and guanidinium isothiocyanate solutions, followed by organic extraction and alcohol precipitation of the RNA.1 TRIzol is the commercial version of this popular single-step method of total RNA isolation. This highly reliable technique performs well with both small and large quantities of tissues or cultured cells and allows simultaneous processing of a large number of samples. The Eppendorf Phase Lock Gel (PLG) separates the aqueous and organic phase with a solid barrier and eases handling. PLG is inert, heat stable and compatible with enzymatic reactions of nucleic acids. The procedure can decrease processing time and improve nucleic acid recovery from organic extraction purifications, all while minimizing user exposure to hazardous organic solutions.2

In this report we describe the use of Eppendorf Phase Lock Gel (PLG) Heavy for the isolation of total RNA from human tissue culture cells and present data regarding the quality, quantity and utility of the RNA recovered.

Materials and Methods

Isolation of total RNA from Jurkat cells

The following protocol was performed to isolate total RNA from cultured eukaryotic cells. The organic extraction of one half of the samples was processed in pre-spun (2030 seconds at 12,000 x g) 2 ml PLG Heavy tubes. The other half was processed in 2 ml standard reaction tubes.

1. Aspirate and discard culture medium from culture vessel.
2. Add 3 ml of TRIzol reagent (Invitrogen) for 2 x107 cells.
3. Incubate homogenized samples for 5 minutes at 15C to 30C.
4. Aliquot lysate into five 2 ml PLG Heavy tubes and five 2 ml standard tubes without PLG (300 ml each).
5. Add 60 l of chloroform/isoamyl alcohol (in a ratio of 49:1, v/v), cover samples tightly and shake vigorously for 15 seconds. Incubate at room temperature for 2 to 3 minutes.
6. Centrifuge samples for 15 minutes at 12,000 x g at 4C.
7. Transfer upper aqueous phase to a fresh tube.
   
8. Precipitate RNA from aqueous phase by mixing with 150 l isopropanol. Store samples at room temperature for 10 minutes and centrifuge at 12,000 x g for 10 minutes. Remove supernatant and wash RNA pellet with 300 l 75% ethanol. Next, centrifuge at 7,500 x g for 5 minutes at room temperature.
9. Remove the ethanol wash and briefly air-dry RNA pellet. Dissolve RNA in 100 l Molecular Biology Grade Water (Eppendorf).

RT-PCR*

1. Heat 3 g of total RNA with 100 ng of oligo-dT-primer for first strand synthesis to 75C for 10 minutes in a reaction volume of 18.5 l. Chill tube on ice for 5 minutes and spin.
2. Add 2.5 l dNTPs (2 mM), 2.5 l 10X RT-buffer and 1.5 l Reverse Transcriptase (300 U) to sample and incubate for 1.5 hours at 42C.
3. Use 2 l of first strand synthesis as template for the PCR amplification of an 844 bp fragment of the human p53 gene.
   Amplification was performed in a final reaction volume of 25 l. PCR was performed with the Eppendorf MasterTaq Kit (50 mM KCl; 10 mM Tris-HCl pH 8.3; 15 mM Mg-Acetate; 1 U Taq DNA Polymerase) and 0.5 M of specific 5 and 3-primers according to the following protocol: The fragments were separated by gel electrophoresis in 1% agarose gel.

Forward primer:
5-ATGGAGGAGCCGCAGTCA3
Reverse primer:
5-CGGTCTCTCCCAGGACAG-3

The PCR reaction was performed on the Eppendorf Mastercycler gradient** using the following cycling conditions:

94C 10 minute initial denaturation
35 cycles:
94C 30 seconds
56C 30 seconds
72C 1 minute
72C 5 minute final extension

Results and Discussion

Determination of yield and quality of the isolated RNA

As shown in Fig.1, the isolation of total RNA using Eppendorf Phase Lock Gel results in greater yields compared to conventional organic extraction preparations. The purity of the prepared RNA is better than without PLG when evaluated with absorbance ratios at A260/280.

Fig. 1: Determination of yield and purity of the isolated total RNA by UV spectrophotometry (Eppendorf BioPhotometer). The average yield of total RNA in g and the average of the ratio A260/280 are shown, comparing the samples purified with PLG and without PLG. Denaturing agarose gel electrophoresis

The most common method used to assess the integrity of total RNA is to run an aliquot of the RNA sample on a denaturing agarose gel stained with ethidium bromide (EtBr). Intact total RNA separated on a denaturing gel will have sharp, clear 28S and 18S rRNA bands (eukaryotic samples). The 28S rRNA bands of the samples used with or without PLG are approximately twice as intense as the 18S rRNA band (Fig. 2). This 2:1 ratio (28S:18S) is a good indication that the RNA is completely intact.

Fig. 2:. Total RNA were separated by gel electrophoresis beside the Gene Ruler DNA Marker 1010,000 bp (Fermentas) on a 1% denaturing agarose gel.

Lane 1: Total RNA prepared with the use of Eppendorf Phase Lock Gel. Lane 2: Total RNA prepared without the use of Eppendorf Phase Lock Gel. Reverse Transcription PCR

In each preparation, an 844 bp fragment of the human p53 cDNA could be amplified from RNA prepared with or without the use of PLG. As shown in Fig. 3, the amplicons are of the same size and approximately the same intensity, and there are no additional bands visible.

Fig. 3: RT-PCR performed on total RNA isolated with TRIzol and Phase Lock Gel. Lanes 15: 844 bp fragment of the human p53 gene amplified by RT-PCR from total RNA prepared with TRIzol and with Eppendorf Phase Lock Gel Lanes 610: 844 bp fragment of the human p53 gene amplified by RT-PCR from total RNA prepared with TRIzol but without Eppendorf Phase Lock Gel Lane 11: Negative control, RT-PCR murine RNA Lane 12: 0.5 mg of DNA size marker (Invitrogen) Range: 506 to 21,216 bp Conclusion

Where it is used as a standard helpful tool for the preparation of DNA via phenol extraction, the Eppendorf Phase Lock Gel (PLG) Heavy can also be used in combination with mono-phasic extraction reagents such as TRIzol for the preparation of total RNA from tissue culture cells. The use of Eppendorf PLG increases the yield of isolated RNA and shortens the preparation time. With the help of PLG the separation of the aqueous and the organic phase is more convenient and reliable and the risk of losing the sample due to organic impurities is minimized (i.e. recovery of the entire aqueous phase is possible).

The RNA is of high purity and not contaminated with proteins or organic solvents measured by UV-absorption. Total RNA prepared with Eppendorf Phase Lock Gel can be directly used for downstream applications sensitive to any kind of impurities in the preparation. Reverse transcription of RNA into cDNA and subsequent amplification could be performed successfully.

In summary, Eppendorf Phase Lock Gel (PLG) Heavy is an excellent tool for simplifying the preparation of total RNA from tissue culture cells using classical organic extraction methods. PLG in combination with TRIzol is an excellent alternative to column-based RNA purification kits.

References

1. Chomcyznski P. and Sacchi N. Anal Biochem. 1987;162, 156.
2. Murphy NR and Hellwig RJ. Biotechniques 1996;21:934.
   

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