Easy Preparation Of Total RNA From Cultured Cells With TRIzol® And Eppendorf® Phase Lock Gel

Easy preparation of total RNA from cultured cells with TRIzol® and Eppendorf® Phase Lock Gel

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 (20–30 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 15°C to 30°C.
4. Aliquot lysate into five 2 ml PLG Heavy tubes and five 2 ml standard tubes without PLG (300 ml each).
5. Add 60 µl 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 4°C.
7. Transfer upper aqueous phase to a fresh tube.
   
8. Precipitate RNA from aqueous phase by mixing with 150 µl 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 µl 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 µl Molecular Biology Grade Water (Eppendorf).

1. Heat 3 µg of total RNA with 100 ng of oligo-dT-primer for first strand synthesis to 75°C for 10 minutes in a reaction volume of 18.5 µl. Chill tube on ice for 5 minutes and spin.
2. Add 2.5 µl dNTPs (2 mM), 2.5 µl 10X RT-buffer and 1.5 µl Reverse Transcriptase (300 U) to sample and incubate for 1.5 hours at 42°C.
3. Use 2 µl 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 µl. 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 µM of specific 5’– and 3’-primers according to the following protocol: The fragments were separated by gel electrophoresis in 1% agarose gel.

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

94°C 10 minute initial denaturation
35 cycles:
94°C 30 seconds
56°C 30 seconds
72°C 1 minute
72°C 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.

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.

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.

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.