mRNA preparation from BY-2

We use combined protocol of total RNA preparation by TRIzolÒ Reagent (Invitrogen 15596-026) and poly (A)+ extraction with GenEluteÔ mRNA Miniprep Kit (Sigma MRN 10 or MRN70).

I. Total RNA preparation

1) Filtrate BY-2 cell culture at desired stage to obtain 1-2 gram of cells on filter paper (note 1). We usually use Buchner funnel and vacuum to remove as much medium from cells as possible. The filtrate that should appear dry is scraped from the filter paper, wrapped into aluminium foil and snap frozen in liquid nitrogen. Cells can be stored in deep freezer (-80 oC) or used directly for RNA preparation.

2) Grind 1-2 gram of frozen cells in liquid nitrogen (note 2). Transfer frozen powdered cells with liquid nitrogen-cooled spatula into 5 ml of TRIzol reagent in 15 ml falcon tube. Shake immediately to suspend cells in TRIzol. Proceed with following samples while keeping already suspended cells at room temperature (note 3).

3) Keep suspensions after processing of the last sample at room temperature for additional 5 minutes and centrifuge homogenate at 12,000 x g for 10 minutes at 4 oC. Transfer cleared homogenate into a fresh tube and add 1 ml of chloroform (0.2 ml chloroform per 1 ml of TRIzol reagent). Cap sample tubes securely and shake vigorously by hand for 15 seconds. Incubate at room temperature for 2-3 minutes.

4) Centrifuge the samples at 10,000 x g at 4 oC for 15 min. Samples will separate into a colorless upper aquaeous phase, a middle protein interphase and a lower organic phase (pink). Collect the upper aquaeous phase with RNA and transfer to a fresh tube. Be careful not to disturb the protein interphase or organic lower phase!

5) Read the actual volume of the collected aquaeous phase and precipitate RNA with 0.8 volume of isopropanol (note 4). Incubate at room temperature for 10 min and centrifugate at 10,000 x g for 10 min at 4 oC.

6) Remove supernatant and wash pellet once with 5 ml of 75% ethanol (1 ml 75% ethanol per 1 ml TRIzol used). Mix samples by vortexing and spin at 7,500 x g for 5 min at 4 oC. Remove all ethanol and air dry RNA for 20-30 minutes in clean bench.

7) Dissolve (note 5) RNA in RNase-free water (e.g. DEPC treated) and store at -70 oC.

8) Measure OD 260/280 and run RNA on agarose gel (note 6) to estimate concentration and quality of total RNA.

II. mRNA preparation

We use TRIzol extracted total RNA for polyadenylated RNA fraction preparation. Sigma produced GenElute mRNA Miniprep Kit provides sufficient amount of high quality mRNA for microarray analysis. We basically follow the manufacturer's protocol in two rounds of subsequent purifications to assure elimination of majority of ribosomal RNAs (note 7). For details refer to original manufacturer's protocol (Sigma MRN10 for 10 preps, MRN70 for 70 preps).

1) Use two RNase-free microcentrifuge tubes to distribute 500 ug of total RNA (note 8) into each of them (total 1mg of RNA is used for each sample purification). Both resulting mRNA fractions will be pooled together for the second round of purification.

Adjust the volume to 250 uL with RNase-free water (provided) and add 250 uL of 2x Binding Solution. Briefly mix by vortexing.

2) Add 15 uL of resuspended oligo (dT) beads and mix thoroughly by vortexing. Incubate at 70 oC for 3 minutes in heating block to denature the secondary structures in RNA.

3) Remove the sample from heating block and allow to stand at room temperature for 10 minutes. Pellet oligo (dT) beads:mRNA complex by centrifuging at maximum speed for 2 min. Carefully remove supernatant and resuspend pellet in 500 uL of Wash Solution by vortexing or pipetting.

4) Transfer the suspension into a GenElute spin filter-collection tube assembly. Spin the whole complex in microcentrifuge at maximum speed for 1 min. Discard the flow through and repeat wash with additional 500 uL of Wash Solution. Centrifuge for 1 minute at max speed and discard flow through.

5) Elute mRNA from the minicolumn with 50 uL of Elution Buffer preheated to 70 oC. Pipet the solution in the middle of filter unit, incubate for 5 minutes at 70 oC and spin for 1 minute at max speed. Repeat elution with another 50 uL of preheated Elution Buffer as previously.

6) Resulting mRNA will now be eluted in total volume of 100 uL of Elution Buffer. This mRNA will still contain substantial amount of ribosomal RNA. To avoid excessive label loss due to ribosomal RNA labeling (in the case of use of random primers for labeling) we re-purify mRNA from two first-round fractions using another single column. To do this, two 100 uL fractions are pooled and volume adjusted to 250 uL with RNase-free water and the whole process of purification is repeated once more.

7) To concentrate the mRNA, a 100 uL of eluted mRNA is finally precipitated in the presence of 0.1 volume of 3M sodium acetate, pH 5.2, and 3 volumes of absolute ethanol and resuspended in 20 uL MiliQ water.

RNA profiles

Total RNA isolated by TRIzol Reagent from BY-2 cell culture

mRNA after one round of poly (A)+ selection on Sigma GenElute column

mRNA after two rounds of poly (A)+ selection on Sigma GenElute column

Protocol notes:

(1) Centrifugation can be used as well for separation of cells from the medium but we find filtration faster and more efficient in removal of excessive medium. Considering the length of centrifugation necessary to pellet the cells and rapid induction of some gene expression, we recommend filtration at this step.

(2) Even though TRIzol can be used to lyze cells directly after filtration or centrifugation, this procedure does not work with BY-2 cells. RNA extracted by this method contains large amounts of tRNA and 5S RNA, but is deficient in longer transcripts including ribosomal RNAs. We assume that direct lysis of BY-2 cells is not sufficient to disintegrate rigid cell walls and only small RNA species are released from partially disrupted cells.

(3) We usually process 6-8 samples at once and according to our experience keeping cells suspended in TRIzol for about 30 minutes at room temperature does not adversely influence the quality of extracted total RNA. For longer periods, the samples should be stored on ice followed by 5 min period at room temperature.

(4) The original protocol uses 0.5 ml of isopropanol to 1 ml of TRIzol used. In the case of BY-2 culture the volume of aquaeous phase varies greatly depending on the content of water in cells and on amount of residual medium after filtration or centrifugation. We rather use 0.8 actual volume of isopropanol to assure good precipitation of RNA.

(5) The usual yield from 1 gram of wet BY-2 cells is about 1mg of total RNA. To dissolve this amount of RNA, at least 300 uL of DEPC treated water is necessary.

(6) In our experience normal 1% agarose gel is sufficient to estimate the quality of RNA. Both ribosomal RNAs should show as strong bands, and also small 5SRNA and tRNA are typically seen at the bottom of the gel.

(7) In our case the contamination with ribosomal RNAs represents 5-10% of the isolated mRNA fraction as measured by Agilent RNA Chip technology.

(8) The amount of total RNA can be scaled down keeping in mind that polyadenylated mRNA fraction represents about 1-2% of total RNA and at least 1ug of mRNA is necessary for labeling and hybridization of a single slide. We usually use 2 microarray slides for each sample and label 2 ug of mRNA in a single labeling reaction with Cy-5 labeled dCTP.