I. Cell Lysis and
Tissue Disruption
1. Collect 102–107
cells or 0.5–250 mg tissue; wash cells
in cold PBS
Cultured mammalian cells
Ideally cells in culture should be
processed fresh (i.e. not frozen). If you need to store cells before RNA
isolation, they can be stored in RNAlater,
or they can be pelleted, frozen in liquid nitrogen,
and stored at –70°C or
colder.
Suspension cells: Count
the cells, then pellet 102–107
cells at low speed, and discard the culture medium. Wash the
cells by resuspending in ~1 ml PBS, and repelleting. Place the washed cells on ice.
Adherent cells: do
one of the following
• Aspirate and discard the culture
medium, and rinse with PBS. Place the culture plate on ice.
• Trypsinize
cells to detach them and count. Then inactivate the trypsin,
pellet the cells, and discard the supernatant (following the method employed in
your lab for the cell type). Wash the cells by gently resuspending
in ~1 ml PBS, and pelleting at low speed. Place the
cells on ice.
Tissue samples
For good yield of intact RNA, it is
very important to obtain tissue quickly and to limit the time between obtaining
tissue samples and inactivating RNases in step c
below.
a. Harvest tissue and remove as much
extraneous material as possible, for example remove adipose tissue from heart,
and remove gall bladder from liver. Perfuse the tissue with cold PBS, if
desired, to eliminate some of the red blood cells.
b. If necessary, quickly cut the tissue
into pieces small enough for either storage or disruption. Weigh the tissue
sample (for samples to be stored in RNAlater,
this can be done later).
c. Inactivate RNases
by one of the following methods:
• Drop the sample into RNAlater—tissue must be cut to ≤0.5
cm in at least one dimension for good penetration of the RNAlater.
• Disrupt the sample in Lysis/Binding Buffer as described in step 2. Fresh
(unfrozen) tissue on page 1.
• Freeze the sample in liquid
nitrogen—tissue pieces must be small enough to freeze in a few seconds. When
the liquid nitrogen stops churning, it indicates that the tissue is completely
frozen. Once frozen, remove the tissue from the liquid nitrogen and store it in
an airtight container at –70°C
or colder.
2. Disrupt samples in 300–600 μl Lysis/Binding Buffer
Cultured cells
a. Remove the PBS wash or the RNAlater, and add 300–600 μl
Lysis/Binding Solution for 100–107
cells. Cells will lyse immediately
upon exposure to the Lysis/Binding Solution. Use the
low end of the range (~300 μl) for small numbers
of cells (hundreds), and use closer to 600 μl
when isolating RNA from larger numbers of cells
(thousands–millions). For adherent
cells lysed directly in the culture plate, collect
the lysate with a rubber spatula, and pipet it into a tube.
b. Vortex or pipet
vigorously to completely lyse the cells and to obtain
a homogenous lysate. Cell cultures typically do not
require mechanical homogenization; however, it will not damage the RNA.
Large frozen cell pellets (i.e. more
than about 107 cells) may
need to be ground to a powder as described for frozen tissue samples to isolate
high quality RNA. Alternatively, samples may be transitioned to –20°C
in RNAlater-ICE (Ambion Cat #7030) and processed as described in step 2.a
above.
c. Proceed to section
Organic Extraction.
Yeast or bacterial cultures
Use disruption techniques appropriate
for yeast and bacterial cultures, which may require vigorous mechanical
disruption.
Solid tissue stored in Ambion’s RNAlater,
or transitioned to –20°C in RNAlater-ICE
Samples in RNAlater
and RNAlater-ICE
can usually be homogenized by following the instructions for fresh tissue
(below). Extremely tough/fibrous tissues in RNAlater
may need to be frozen and pulverized
according to the instructions for frozen tissue in order to achieve good cell
disruption. Blot excess liquid from samples, and weigh them before following
the instructions for fresh tissue below.
Fresh (unfrozen) tissue
Use at least a 1:10 ratio (w/v) of tissue
to Lysis/Binding Buffer for all tissues.
a. Measure or estimate the weight of
the sample.
b. Aliquot 10 volumes per tissue mass
of Lysis/Binding Buffer into a homogenization vessel
on ice (e.g. aliquot ml Lysis/Binding Buffer for 0.1
g tissue). Keeping the sample cold, thoroughly disrupt the tissue in Lysis/Binding Buffer using a motorized rotor-stator
homogenizer. A ground-glass homogenizer or a plastic pestle can be used for
small pieces (≤10 mg) of
soft tissue. Homogenize until all visible clumps are dispersed.
c. Proceed to section II.E.
Organic Extraction on page 11.
Frozen tissue, and extremely hard
tissues
(Frozen tissue transitioned to –20°C in
RNAlater-ICE: process as for fresh tissue).
Once the tissue has been removed from the –70°C
freezer, it is important to process it immediately without even partial
thawing.
This is necessary because as cells
thaw, ice crystals rupture both interior and exterior cellular compartments,
releasing RNases.
a. Measure or estimate the weight of
the sample.
b. Place 10 volumes of Lysis/Binding Buffer per tissue mass into a plastic weigh
boat or tube on ice. We suggest using a weigh boat because it is easier to
transfer frozen powdered tissue to a weigh boat than to a tube of Lysis/Binding Buffer.
c. Grind frozen tissue to a powder with
liquid nitrogen in a prechilled mortar and pestle
sitting in a bed of dry ice.
d. Using a prechilled
metal spatula, scrape the powdered tissue into the Lysis/Binding
Buffer, and mix rapidly.
e. Transfer the mixture to a vessel for
homogenization and process the mixture to homogeneity, i.e. until all visible
clumps are dispersed. If available, use a motorized rotor-stator homogenizer
(e.g. Polytron).
f.
Proceed to Organic Extraction below.
II. Organic Extraction
Include this organic extraction both
for total RNA purification and for purification of small RNAs.
1. Add 1/10 volume of miRNA Homogenate Additive, incubate 10 min on ice
a. Add 1/10 volume of miRNA Homogenate Additive to the cell or tissue lysate
(or homogenate), and mix well by vortexing or
inverting the tube
several times. For example, if the lysate volume is
300 μl, add 30
μl miRNA Homogenate
Additive.
b. Leave the mixture on ice for 10 min.
2. Extract with a volume of Acid-Phenol:
Chloroform equal to the initial lysate volume
a. Add a volume of Acid-Phenol:Chloroform that is equal to the lysate
volume before addition of the miRNA Homogenate
Additive. For example, if the original lysate volume
was 300 μl, add 300 μl
Acid Phenol:Chloroform.
b. Vortex for 30–60 sec to mix.
c. Centrifuge for 5 min at maximum
speed (10,000 x g)
at room temperature to separate the aqueous and organic phases. After
centrifugation, the interphase should be compact; if
it is not, repeat the centrifugation.
3. Recover the aqueous phase; transfer
the aqueous phase to a fresh tube
Carefully remove the aqueous (upper)
phase without disturbing the lower phase, and transfer it to a fresh tube. Note
the volume removed.
III. Final RNA Isolation
A.Total
RNA Isolation Procedure
1. Add 1.25 volumes 100% ethanol, and
mix thoroughly
Add 1.25 volumes of room temperature
100% ethanol to the aqueous phase (e.g. if 300 μl
was recovered in step E.3, add 375 μl ethanol).
2. Pass the lysate/ethanol
mixture through a Filter Cartridge
a. For each sample, place a Filter
Cartridge into one of the Collection Tubes supplied.
b. Pipet the lysate/ethanol mixture (from the previous step) onto the
Filter Cartridge. Up to 700 μl can be applied to
a Filter Cartridge at a time, for samples larger than this, apply the mixture
in successive applications to the same filter.
c. Centrifuge for ~15 sec to pass the
mixture through the filter. Centrifuge at RCF 10,000 x g (typically 10,000 rpm). Spinning
harder than this may damage the filters.
Alternatively, vacuum
pressure may be used to pass samples through the filter.
d. Discard the
flow-through, and repeat until all of the lysate/ethanol
mixture is through the filter. Reuse the Collection Tube for the washing steps.
3. Wash the filter with 700 μl
miRNA
Apply 700 μl miRNA Wash Solution 1
(working solution mixed with ethanol) to the Filter Cartridge and centrifuge
for ~5–10 sec or use a vacuum to pull the solution through the filter. Discard
the flow-through
from the Collection Tube,
and replace the Filter Cartridge into the same Collection Tube.
4. Wash the filter twice
with 500 μl
a. Apply 500 μl Wash Solution 2/3 (working solution mixed with ethanol)
and draw it through the Filter Cartridge as in the previous step.
b. Repeat with a second 500
μl aliquot of Wash Solution 2/3.
c. After discarding the
flow-through from the last wash, replace the Filter Cartridge in the same
Collection Tube and spin the assembly for 1 min to remove residual fluid from
the filter.
5. Elute RNA with 100 μl 95°C Elution Solution or Nuclease-free Water
Transfer the Filter
Cartridge into a fresh Collection Tube (provided with the kit). Apply 100 μl of pre-heated (95°C) Elution Solution or nuclease-free
water to the center of the filter, and close the cap. Spin for ~20–30 sec at
maximum speed to recover the RNA. Collect the eluate
(which contains the RNA) and store it at –20°C or
below.
B. Enrichment Procedure for
Small RNAs
This variation of a
traditional glass-fiber filter RNA purification yields RNA that is
significantly enriched for small RNAs. This
enrichment is accomplished by first immobilizing large RNAs
on the filter with a relatively
low ethanol concentration
and collecting the flow-through containing mostly small RNA species. More
ethanol is then added to this flow-through, and the mixture is passed through a
second glass filter where the small RNAs are
immobilized. This second filter is then washed a few times, and the small-RNA
enriched sample is eluted.
1. Add 1/3 volume 100%
ethanol, and mix thoroughly
Add 1/3 volume of 100%
ethanol to the aqueous phase recovered from the organic extraction (e.g. add
100 μl 100% ethanol to 300 μl
aqueous phase). Mix thoroughly by vortexing or
inverting the tube several times.
2. Pass the sample through a
Filter Cartridge, and collect the filtrate
a. For each sample, place a
Filter Cartridge into one of the Collection Tubes supplied.
b. Pipet
the lysate/ethanol mixture (from the previous step)
onto the Filter Cartridge. Up to 700 μl can be
applied to a Filter Cartridge at a time. For sample volumes greater than 700 μl, apply the mixture in successive applications to
the same filter.
Collect the filtrate (that is, the liquid flow-through in the Collection Tube); it
contains the small RNAs.
c. Centrifuge for ~15 sec
to pass the mixture through the filter. Centrifuge at RCF 10,000 x g (typically 10,000 rpm). Spinning harder than this may damage the
filters. Alternatively, vacuum pressure can be used to pull samples through the
filter.
d. Collect the filtrate. If
the lysate/ethanol mixture is >700 μl, transfer the flow-through to a fresh tube, and repeat until all of the lysate/ethanol
mixture is through the filter. Pool the collected filtrates if multiple passes
were done, and measure the total volume of the filtrate.
NOTE
At this point, the filter
contains an RNA fraction that is depleted of small RNAs.
This fraction can be recovered if desired by washing and eluting from the filter as described
in steps F.I.3–5.
3. Add 2/3 volume 100%
ethanol and mix thoroughly
Add 2/3 volume room
temperature 100% ethanol to filtrate (i.e. flow-through). For example, if 400 μl of filtrate is recovered, add 266 μl 100% ethanol. Mix thoroughly.
4. Pass the mixture through a
second Filter Cartridge, and discard the flow-through
a. For each sample, place a
Filter Cartridge into one of the Collection Tubes supplied.
b. Pipet
the filtrate/ethanol mixture (from the previous step) onto a second Filter
Cartridge. Up to 700 μl can be applied to a
Filter Cartridge at a time. For sample volumes greater than 700 μl, apply the mixture in successive applications to
the same filter.
c. Centrifuge for ~15 sec
to pass the mixture through the filter. Centrifuge at RCF 10,000 x g (typically 10,000 rpm). Spinning harder than this may damage the
filters. Alternatively, vacuum may be used to pass samples through the filter.
d. Discard the flow-through,
and repeat until all of the filtrate/ethanol mixture is through the filter.
Reuse the Collection Tube for the washing steps.
15
5. Wash the filter with 700 μl miRNA
Apply 700 μl miRNA Wash Solution 1
(working solution mixed with ethanol) to the Filter Cartridge and centrifuge
for ~5–10 sec or use vacuum to pass the solution through the filter. Discard
the flow-through from the Collection Tube, and replace the Filter Cartridge
into the same Collection Tube.
6. Wash the filter twice
with 500 μl
a. Apply 500 μl Wash Solution 2/3 (working solution mixed with ethanol)
and draw it through the Filter Cartridge as in the previous step.
b. Repeat with a second 500
μl aliquot of Wash Solution 2/3.
c. After discarding the
flow-through from the last wash, replace the Filter Cartridge in the same
Collection Tube and spin the assembly for 1 min to remove residual fluid from
the filter.
7. Elute RNA with 100 μl 95°C Elution Solution or Nuclease-free Water
Transfer the Filter
Cartridge into a fresh Collection Tube (provided with the kit). Apply 100 μl of pre-heated (95°C) Elution Solution or nuclease-free
water to the center of the filter, and close the cap. Spin for ~20–30 sec at
maximum speed to recover the RNA. Collect the eluate
(which contains the RNA) and store it at –20°C or colder.
IV. Analyzing RNA after
Purification RNA
quantitation and quality assessment
Measuring the absorbance at
260 nm (A260) in a spectrophotometer is a simple method for determining the
concentration of RNA. The ratio of A260 to A280 provides an indication of
RNA purity. See section IV.B on
page 22 for detailed
instructions on using spectrophotometer readings to assess RNA. An alternative
way to estimate RNA quality and quantity is to run a sample on a denaturing
gel. To visualize small RNAs, use a denaturing acrylamide gel. To visualize ribosomal RNA, run the samples
in a denaturing agarose gel.
Part II. miRNA
Labeling and Clean-Up
A. Input RNA Requirements
Obtaining sample miRNA
The first step in miRNA analysis using the mirVana Array System is to obtain miRNA. The
two principal ways to obtain miRNA are to purify RNA
using a technique that enriches for the small RNA fraction, (e.g., using Ambion’s mir Vana miRNA Isolation Kits), or to
isolate total RNA containing the small RNA fraction, and then to physically
separate the miRNA fraction from total RNA using gel
electrophoresis (e.g., with Ambion’s flashPAGE Fractionator). See the
detailed instructions below. Although the expression profiles obtained from
enriched and flashPAGE-purified miRNAs
are very similar, we recommend using flashPAGE-purified
miRNA samples for array analysis because it results
in higher signal and lower background compared to results using samples
obtained with a miRNA enrichment procedure alone. In
addition, we found that using flashPAGE-purified miRNA for array analysis provides better sensitivity,
accuracy, and reproducibility than using total
RNA or the
small-RNA-enriched fraction.
Note: ~0.01% of total RNA is miRNA. flashPAGE purification achieves ~10,000 fold enrichment for
miRNA,
whereas enrichment using the mirVana RNA isolation kits results in ~10 fold enrichment
for miRNA and
other small RNAs.
Recommended: flashPAGE miRNA
We recommend labeling the miRNA purified from 5–20 μg
of total RNA using Ambion’s flashPAGE
Fractionator for each array hybridization.
• Isolate or purchase total
RNA that includes the full complement of miRNAs. Typically,
RNA isolation methods that use RNA-binding glass-fiber filters do not
quantitatively recover RNA species <200 nt. We recommend
using Ambion’s
mirVana miRNA
Isolation Kit (Cat #1560) or mir Vana PARIS™ Kit (Cat #1556) to purify total
RNA or to isolate RNA
fractions enriched for small RNA species.
Alternative: miRNA-enriched fraction of total RNA
For preliminary experiments
or experiments to identify highly expressed miRNAs,
labeling RNA enriched for the small RNA fraction may be sufficient. Use Ambion’s mir Vana miRNA Isolation Kit (Cat
#1560) or mir Vana PARIS™ Kit (Cat #1556), and follow
the small RNA enrichment protocol.
Suggested starting total RNA
mass
We recommend labeling miRNA obtained from 5–20 μg
of total RNA for analysis with mirVana miRNA Bioarrays
or with arrays made using the mirVana miRNA Probe Set. We recommend a broad
range of input
miRNA because with some targets,
saturation of signal may be seen with labeled miRNA
obtained from only 20 μg of total RNA, whereas
other targets may be difficult to detect even with labeled miRNA
derived from 50 μg of total RNA. The relative
mass of miRNAs varies considerably among different
sample types. For example, 10 μg of total RNA
from most tissues contains approximately 1 ng of miRNA, whereas 10 μg of total
RNA from cultured cells contains only ~250 pg of miRNA.
For total RNA samples that contain relatively little miRNA,
label miRNA derived from up to 50 μg
of total RNA for array hybridization. Conversely, for hybridization to
mir Vana miRNA
Bioarrays with samples that contain a relatively
high miRNA content, labeled miRNA
derived from as little as
1–2 μg of total RNA may be sufficient.
B. Appending 3' Amine-Modified Tails to miRNA
Before you start:
• Dry miRNA
samples derived from 5–20 μg of total RNA to
completion by vacuum centrifugation.
• Warm non-enzyme reaction components
to room temp.Remove the non-enzyme reagents that will
be used in the Poly(A) Polymerase reaction (step 3 below) from the freezer, and
allow them to equilibrate to room temp for 2 hours. [Leave the Poly(A)
Polymerase at –20°C.]
• Preheat a 37°C incubator.
1. Dilute the Positive
Control miRNA in Nuclease-free Water
The Positive Control miRNA samples supplied with the mirVana miRNA Labeling Kit and the mirVana miRNA Bioarray
Essentials Kit(Cat #1566) are identical. The control is designed to hybridize
with the mir Vana miRNA Bioarrays Control_1 probe (BA10001) and the mir- Vana
miRNA Probe Set Control_1 probe (PS20001).
For mirVana miRNA Bioarrays, dilute 1 μl of Positive Control miRNA
with 99 μl Nuclease-free Water.
To prepare samples for analysis
on mirVana miRNA
Bioarrays, dilute 1 μl
of the Positive Control miRNA into 99 μl Nuclease-free Water. The Positive Control miRNA must
be added to each miRNA sample that will be hybridized with mirVana miRNA
Bioarrays; it serves as a positive control for sample
labeling and hybridization, and also produces fiducial
spots that are recognized by array scanning equipment and are necessary for
array analysis.
For mirVana miRNA Probe Set arrays, dilute 1 μl
of Positive Control miRNA with 499 μl Nuclease-free Water.
(optional) For mirVana miRNA
Probe Set arrays, dilute 1 μl of the Positive Control
miRNA with 499 μl
Nuclease-free Water. The Positive Control miRNA can
be added to each sample miRNA as a spike control for
samples that will be hybridized with miRNA arrays made
using the mirVana miRNA
Probe Set.
Store the diluted Positive
Control miRNA in small aliquots at –70°C between
experiments.
2. Resuspend
miRNA samples in 3 μl Nuclease-free
Water
(Recommended) miRNA isolated using the flashPAGE
Fractionator (or gel electrophoresis): use the miRNA
fraction obtained from 5–20 μg of total RNA. (See section II.A for details.) RNA enriched for miRNA:
(obtained with the mirVana miRNA
Isolation Kit or the mirVana PARIS Kit): use 0.5–5 μg enriched miRNA or the miRNA
derived from 5–20 μg total RNA. Add 3 μl Nuclease-free Water to
each dried miRNA sample, and resuspend the miRNA by vortexing
briefly and/or by pipetting up and down a few times.
3. Add Poly(A) Polymerase reaction
mix to each miRNA sample
At room temp, add the
tailing reaction reagents to each miRNA sample in the
order shown below and mix well by gently flicking the tube a few times. For
experiments that include >2 samples, it is a good idea to prepare a master mix.
1 μl diluted Positive Control miRNA*
* If you are preparing sample for analysis on an miRNA array prepared using the mirVana miRNA Probe Set, and you choose not to
include the Positive Control miRNA in your reaction,
then replace it with Nuclease-free Water.
10 μl 2X Poly(A) Polymerase Reaction Buffer
2 μl 25 mM MnCl2
2 μl 10X Amine-NTP Mix
2 μl Poly(A) Polymerase
4. Incubate at 37°C for 2 hr
Incubate the tailing
reaction at 37°C for 2 hr.
NOTE :Begin heating the Column Elution Buffer to 95°C towards the end of
this incubation.
It will be used in step C.5
below.
11
C. Post-Tailing miRNA Clean-Up
This clean-up procedure
removes unincorporated nucleotides from the tailed miRNA
by filter purification with the miRNA Labeling
Columns.
Before you start:
• Add 38 ml ACS-grade or
higher quality 100% ethanol to the bottle of miRNA
Binding/Wash Buffer, mix well, and mark the label to indicate that the ethanol
was added.
• Preheat the Elution
Solution to 95°C
• Preheat an incubator to
65°C
1. To each reaction add the following
and incubate 5 min at room temp:
a. Add miRNA
Carrier and miRNA Binding/Wash Buffer to each sample
according to the table below.
Make sure that the ethanol
has been added to the miRNA Binding/ Wash Buffer
before use.
10 μl
miRNA Carrier
350 μl
miRNA Binding/Wash Buffer
b. Mix by vortexing briefly, then incubate at room temp for 5 min.
2. Pass mixture through an miRNA Labeling Cartridge
a. Pipet
the mixture from the previous step into an miRNA
Labeling Cartridge (in an miRNA Collection Tube) and
centrifuge at ~10,000 x g for 15 sec, or until the mixture is through the filter.
b. Discard the flow-through
and place the miRNA Labeling Cartridge back in the
Collection Tube.
3. Wash with 2 x 300
μl miRNA Binding/Wash Buffer
a. Pipet
300 μl miRNA
Binding/Wash Buffer into the miRNA Labeling Cartridge
and centrifuge at ~10,000 x g for 15 sec, or until the buffer is through the filter.
b. Discard the flow-through
and place the miRNA Labeling Cartridge back in the
Collection Tube.
c. Repeat steps a–b to wash
with a second 300 μl of miRNA
Binding/ Wash Buffer.
4. Dry the filter Dry the filter by centrifuging at ~10,000 x g for 1 min.
Amount Component
5. Elute tailed miRNA with 30 μl hot Elution
Solution
a. Transfer the cartridge
to a fresh miRNA Collection Tube.
b. Add 15 μl of 95°C Elution Solution to the miRNA
Labeling Cartridge and incubate at 65°C for 5–10 min.
c. Centrifuge at ~10,000 x g briefly (just until the centrifuge comes up to speed) to collect
the tailed miRNA in the tube.
d. With the miRNA Labeling Cartridge still in the tube, add a second 15
μl of 95°C Elution Solution, and incubate at
65°C for 5–10 min.
e. Centrifuge at ~10,000 x g for 1 min to collect all of the tailed miRNA
in a single tube.
6. Dry samples in a vacuum concentrator
Dry samples to completion
in a vacuum concentrator. (If desired the dried sample can be stored at –20°C for 1–2 days).
D. Labeling the Tailed miRNA with Amine-Reactive CyDye
Amine-modified miRNA samples prepared with this kit can be labeled with
any of a number of detectable moieties. We have used Cy5 and Cy3 extensively at
Ambion, and we recommend them for array experiments.
We have also labeled with Alexa dyes and found them to work well. Similar fluorescent
dyes and detectable moieties such as digoxigenin and
biotin are also expected to be compatible with this procedure. Most
amine-reactive dyes are provided in convenient sizes to facilitate nucleic acid
labeling. Here we provide instructions for using Cy3, Cy5, and Alexa Fluor dyes. For other
labeling moieties, follow the manufacturer’s recommendation to resuspend and aliquot the dye prior to use.
1. Resuspend
in 7 μl Nuclease-free Water
Add 7 μl
Nuclease-free Water to each dried sample and vortex briefly to resuspend the tailed miRNA.
2. Resuspend
Cy3- or Cy5-NHS ester with 16 μl of DMSO
CyDyes: Resuspend one vial
of Cy3 or Cy5 Post Labelling Reactive Dye (Amersham Biosciences) with 16 μl
of DMSO. Alexa Fluor dyes: Resuspend Alexa Fluor
dyes in 4 μl DMSO.
• Prepare dye immediately
before starting the dye coupling procedure.
• It is very important that
dye compounds remain dry both before and after dissolving in DMSO, because any introduced
water will cause hydrolysis of the NHS esters, lowering the efficiency of
coupling.
• Store any unused solubilized dye in the dark at –80°C. Note that Amersham Biosciences reports that solubilized
dye has a very limited shelf life.
3. Add 9 μl
Coupling Buffer and 4 μl prepared CyDye to each sample
Set up each labeling
reaction as follows: Vortex briefly to mix, and shield from light as much as
possible.
9 μl
Coupling Buffer
7 μl
amine-modified miRNA
4 μl
prepared Cy3 or Cy5 in DMSO
4. Incubate 1 hr at room temp
in the dark
This 1 hr incubation at
room temp allows the dye coupling reaction to occur. To keep the samples in the
dark, simply put the tubes in a closed drawer.
5. Add 4.5 μl 4M Hydroxylamine and mix
To quench the reaction, add
4.5 μl 4M Hydroxylamine and mix well by vortexing gently.
6. Incubate 15 min at room temp
in the dark
Incubate the reaction in the
dark at room temp for 15 min. During this incubation, the large molar excess of
hydroxylamine will quench the amine-reactive groups on the unreacted
dye molecules.
E. Post-Labeling miRNA Clean-Up
IMPORTANT: Keep your sample in the dark as much as possible to avoid photobleaching.
Before you start:
• Preheat the Nuclease-free
Water to 95°C
• 65°C incubator needed
1. Add 350 μl miRNABinding/Wash Buffer
and incubate 5 min at room temp in the dark
a. Add 350 μl miRNA Binding/Wash Buffer
to each labeled miRNA reaction and vortex briefly to
mix thoroughly.
b. Incubate at room temp in
the dark for 5 min.
2. Pass mixture through an miRNA Labeling Cartridge
a. Pipet
the mixture from the previous step into the miRNA
Labeling Cartridge (in an miRNA Collection Tube) and
centrifuge at ~10,000 x g for 15 sec, or until the mixture is through the filter.
b. If you plan to cohybridize two samples labeled with different dyes to a
single array: add the second sample to the same miRNA
Labeling Cartridge as the first sample, and centrifuge as in the previous step.
3. Wash with 2 x 300
μl miRNA Binding/Wash Buffer
a. Pipet
300 μl miRNA Binding/Wash
Buffer into the miRNA Labeling Cartridge and
centrifuge at ~10,000 x g for 15 sec, or until the buffer is through the filter.
b. Discard the flow-through
and place the miRNA Labeling Cartridge back in the
Collection Tube.
c. Repeat steps a–b to wash
with a second 300 μl miRNA
Binding/ Wash Buffer.
4. Dry the filter
Dry the filter by
centrifuging at ~10,000 x g for 1 min.
5. Elute tailed miRNA with 22 μl 95°C
Nuclease-free Water and 10 min incubation at 65°C
a. Transfer the column to a
fresh miRNA Collection Tube
b. Add 22 μl of 95°C Nuclease-free Water the miRNA
Labeling Cartridge and incubate at 65°C for 10 min.
c. Centrifuge at ~10,000 x g for 1 min to collect the labeled miRNA
in the tube.
6. Immediately proceed to array
hybridization
It is important to use the
labeled sample for miRNA array hybridization within
30 min after elution from the cartridge.
15
Part III. microRNA Array Hybridization, washing and scanning
1. Preheat 3X miRNA
Hybridization Buffer to 65°C for >5 min
Heat the 3X miRNA
Hybridization Buffer to 65°C for at least 5 min immediately before use. Vortex
the tube a few times at maximum speed for ~15 sec during this preheating
incubation. Even after heating and vortexing, you may
see precipitate in the bottom of the tube; it will not affect hybridization.
Keep the solution at 65°C until use.
2. Clean a Bioarray
LifterSlip™ and place it over the Bioarray
a. Clean a Bioarray
LifterSlip coverslip with
70% ethanol, and wipe it dry with a lint-free laboratory wipe. (Ordinary
low-lint laboratory wipes can be used, but they will deposit some dust
particles that must be removed in the next step.)
b. Using the same wipe, remove all dust
particles from the coverslip.
c. Examine the Bioarray
LifterSlip to determine on which side the raised
edges are located. We do this by carefully examining the reflection of overhead
lights on the glass; the white strips that “lift” the coverslip
away from the slide appear dull because they do not reflect the light. Using
Figure 2 as a guide, gently place the Bioarray LifterSlip (raised-edge down) over both miRNA
Bioarrays on the slide so that the raised edges align
with the long edges of the slide, and with the alignment marks on the slide.
3. Assemble the miRNA
hybridization mixture with labeled miRNA and 1X final
concentration miRNA Hybridization Buffer to cover the
array
a. Using sample prepared with the mirVana
miRNA Labeling Kit, bring the volume of the labeled
sample to 20 μl with Nuclease-free Water.
b. Add 10 μl
preheated 3X miRNA
Hybridization Buffer and mix thoroughly. Using the Bioarray
LifterSlips provided in the mirVana
miRNA Bioarray Essentials
Kit, ~30 μl of miRNA
hybridization mixture will completely fill the space under the coverslip.
IMPORTANT: It is
important to prepare enough miRNA hybridization
mixture to completely fill the area
under the coverslip. Also, the final concentration of
miRNA
Hybridization Buffer in the mixture must be 1X for optimal
hybridization.
4. Heat to 95°C for 2 min, then let the
mixture cool to room temp
a. Heat the miRNA
hybridization mixture to 95°C for 2 min.
b. Briefly centrifuge to bring the
sample to the bottom of the tube, and leave the mixture in the dark for ~1 min
to allow it to cool to room temperature.
5. Hybridize for 12–16 hr at 42°C
a. Place the array slide(s) into a
hybridization chamber (e.g., Corning® Product
#2551).
b. Position the pipette tip at an open
side of the coverslip and slowly pipette the entire miRNA hybridization mixture under the Bioarray
LifterSlip onto the mirVana
miRNA Bioarray (see Figure
3). Do not reposition the pipette tip while you are dispensing the miRNA hybridization mixture as this may result in a trapped
bubble.
c. Add 1X
Hybridization Buffer (dilute the 3X Hybridization Buffer to a 1X concentration with nuclease-free water)
to the designated places in the chamber to maintain humidity during
hybridization. This is critical for avoiding problems caused by drying out of
the miRNA hybridization mixture.
d. Check the position of the Bioarray LifterSlips, and gently
push them back into the alignment marks if they have drifted. Then seal the
hybridization chamber completely. Keep the
hybridization chamber in the
dark during hybridization to avoid photobleaching.
e. Place the sealed hybridization
chamber in a 42°C waterbath (resting on the floor of
the waterbath) and incubate for 12–16 hr. If the waterbath has a clear cover, or a hole for a thermometer,
cover the lid to prevent light from entering the waterbath
during hybridization.
II. Washing
IMPORTANT: During
the wash procedure, carefully avoid exposing the slides to air for more
than 1–2 sec. Longer exposures typically result in drying,
which causes the formation of
precipitates on the array. It is also very important to complete the
wash procedure quickly (i.e., in 15 min or less), and in as
little light as possible (to
avoid photobleaching of the labeled sample).
1. Prepare array wash solutions
• Assemble 2 slide holders and 3 slide
wash containers, and determine the volume of wash solution needed to fill the
containers so that the slides in a slide holder will be completely immersed in
solution.
• You will need 1 container
of Low Stringency Wash, and two containers of High Stringency Wash.
• Starting with the
nuclease-free water, assemble the wash solutions and mix thoroughly.
High strangnecy
wash buffer
376 ml
Nuclease-free Water
4 ml Detergent
Concentrate
20 ml
Salt Concentrate
Low strangnecy
wash buffer
780 ml
Nuclease-free Water
0 ml Detergent
Concentrate
20 ml
Salt Concentrate
2. Submerge miRNA Bioarray in Low Stringency
a. Remove the hybridized miRNA Bioarray slide from the
hybridization chamber and submerge it in Low Stringency Wash at room temp. The coverslip will disengage from the slide and fall to the
bottom of container. Quickly place the slide in a slide rack in the wash
container.
b. Leave the slide in the
Low Stringency Wash, and wash at room temp for ~30 sec by dipping the slide
rack up and down in the container of wash solution or with gentle stirring
(150–200 rpm).
3. Change slide rack and
wash in High Stringency
a. Transfer the miRNA Bioarray to a clean slide
rack that is already submerged in the High Stringency Wash, being careful not
to expose the array to the air for more than 1–2 seconds.
b. Wash in High Stringency
Wash at room temp for 30 sec by dipping the slide rack up and down in the
container of wash solution or with gentle stirring (150–200 rpm).
4. Wash in fresh High
Stringency
Transfer the slide rack
with the miRNA Bioarray to
a clean container of fresh High Stringency Wash, and wash for 30 sec by dipping
the slide holder up and down in the container of wash solution or with gentle
stirring (150–200 rpm).
5. Centrifuge the slide to
dry it
a. Rapidly transfer the miRNA Bioarray slide to a
centrifuge equipped with a slide holder. Alternatively, the entire slide rack
can be centrifuged in a microtiter plate rotor
adapter.
b. Start the centrifugation
immediately after transferring the slides to the centrifuge. At Ambion, we typically cover the top of the centrifuge to
keep out the light.
• In a picofuge,
centrifuge for 1–2 min (speed is not adjustable).
• In a tabletop centrifuge,
spin at 600 x g for 3 min.
III. Image Acquisition
Digital images are made up
of many small square pixels, each with its own intensity value. The resolution
of an image is the total number of pixels and is expressed as the number of
rows and columns of pixels in the image (e.g., 1600 x 1200). The number of possible
values for an individual pixel is determined by the color depth; 16 bits/pixel
is typical for array analysis systems. This means that individual pixels can
have intensity values ranging from 0 to 65,535. Pixels exhibiting the maximum intensity
value or higher are “saturated” and are assigned the intensity value 65,535. In
other words, all signals at the maximum intensity value and higher are assigned
the same value. Scanning is the process of illuminating a microarray with laser
light and collecting the light emitted by bound fluorescent dye. The laser is
set to emit the wavelength that excites the fluorescent dye used to label the sample,
and the resulting fluorescence is then measured. Any of a number of
commercially available scanners can be used to collect and analyze microarray
data. Ambion uses the Axon® GenePix 4000B
scanner and associated GenePix software.
Orientation of the mirVana miRNA Bioarrays on the slide
In a low resolution scan, mirVana miRNA
Bioarrays will be visible as a small square in the
middle of the area covered by the coverslip. By zooming
in on the array, the positive control spots should be visible in the first and last
row. There are two additional control spots on the bottom left of the image.
Scan settings for mirVana miRNA Bioarrays
Scan mirVana miRNA
Bioarrays at a photomultiplier tube (PMT) setting of
600 with 100% power and 5 μm resolution. Do not
adjust these setting for individual arrays.
IV. Data analysis