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
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
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
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.
Total RNA isolated by TRIzol Reagent from BY-2 cell culture
mRNA after one round of poly (A)+ selection on Sigma GenElute
mRNA after two rounds of poly (A)+ selection on Sigma GenElute
(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
(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
(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
(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.