10 protocols
AccessionNameType
P-MTAB-30117
hybridization
Hybridization of 70mer oligonucleotide microarrays using the HS4800 hybridization station

(Tecan) in conjunction with manual washing



• start the HS4800 Control Manager Software

• if a dialog box for setting up a new HS Control Manger license appears click on “Cancel”

• a login window appears

• enter login and password

• switch on the hybridization machine

• insert tubings into the washing solutions

• insert slide adapters with dummy slides into the hybridization modules to be used (always

insert 4 dummy slides per module)

• check if screws closing the injection ports are really tightend

• click on to connect the instrument to the computer

• click on to switch on the heating of the Liquid Distribution Unit

• open an appropriate hybridization program

• never change programs yourself, ask personnel of the Transcriptomics Facility for help

• select the hybridization chambers to be used by clicking on

• click on P to prime the channels to be used

• select the channels to be used and prime each for 30 sec (channels can only be primed

separately)

• insert slide adapters with microarrays into the hybridization modules (always insert 4 slides

per module, if you have less microarrays use additional dummy slides)

• click on go to start the program

• wait until a notification window “Preparing probe injection” appears (an acustic signal

reminds you of injecting the samples)

• press OK on the control panel of the instrument

• open the injection port of the first hybridization chamber to be used

• inject the first sample (100 ul) using a pipetman (do NOT use filter tips)

• close the injection port

• press OK on the control panel of the instrument

• open the injection port of the second hybridization chamber to be used

• inject the second sample, press OK on the control panel of the instrument

• close the injection port

• proceed with the remaining samples following the same procedure until all samples have

been injected

• after the last confirmation of injection by pressing OK the program continues automatically

with the hybridization step

• prepare 250 ml or 500 ml, respectively (250 ml are needed for each individual washing step

listed below), of the washing buffers in demineralized water from appropriate stocks

(20×SSC, 10 %(w/v) SDS), preheat 2×SSC, 0.2 %(w/v) SDS washing buffer to 42 degrees C and

cool the 0.05×SSC washing buffer to 18 degrees C

• prepare 2 black plastic boxes and one black plastic slide rack. Also prepare an 12x8 cm petri

dish (Genomics Solutions) containing 1 Kim-wipe and stacks of three used slides each at the

sides

• just before the program terminates, pour 250 ml 2×SSC, 0.2 %(w/v) SDS washing buffer

prewarmed to 42 degrees C in 1 black plastic box

• wearing gloves, remove the slide adapters one by one from the hybridization modules (do

not touch the DNA side of the slide, only touch the edges of the slide!), quickly remove the

slides one by one from the adapter and place them into the black plastic slide rack that is

immersed in the first container in the prewarmed 2×SSC, 0.2 %(w/v) SDS washing buffer

• move up and down several times immediately and shake for 1 min on a horizontal shaker

after removal of the last slide. From now on regularly move slide racks up and down to avoid

air bubble formation

• proceed with the following washing steps:

- transfer to 0.2×SSC, 0.1 %(w/v) SDS (RT, at most 24 degrees C) in a black plastic rack, shake for

1 min

- transfer to 0.2×SSC, 0.1 %(w/v) SDS (RT, at most 24 degrees C) in a black plastic rack, shake for

1 min

- transfer to 0.2×SSC (RT, at most 24 degrees C) in a black plastic rack, shake for 1 min

- transfer to 0.2×SSC (RT, at most 24 degrees C) in a black plastic rack, shake for 1 min

- transfer to 0.05×SSC (Important: set to 18 degrees C and remove from the cooler

immediately before use) in a black plastic rack, shake for 1 min

• place rack on an 12x8 cm plastic microplate cover (Genomics Solutions) containing 2 Kimwipes

and immediately centrifuge in the microplate centrifuge at 1.200 rpm for 3-5 min. Use

a stack of 3 used glass slides at every side of the plastic dish to lift up the rack with the

slides, this avoids precipitation artetacts at the side of the slide. Be sure to counter-balance

using an appropriate balance. If necessary, dry corners of the slide afterwards with a Kimwipe

• place dried slides in a box in the dark until scanning. This may avoid bleaching effects in the

Cy5 channel. Work quickly at all times, avoid direct light and exposure to high ozone

concentrations, since this strongly enhances Cy5-bleaching.

• insert slide adapters with dummy slides in all modules that were used (always insert 4 slides

per module, even if less than 4 slides have been used for hybridization)

• click on to switch off the heating of the Liquid Distribution Unit

• insert tubings of all channels that have been used into a 2 l bucket filled with filtered and

autoclaved MilliQ wate

• click on R to rinse the channels that have been used

• only single selected channels can be rinsed at once or all 6 channels of the instrument can

be rinsed in parallel

• remove all tubings from the solutions

• open the nitrogen gas supply

• open the program “Final drying” and select all slides of each module that has been used for

hybridization

• start the program

• after the drying program has finished remove the adapters from all modules that have been

used

• clean the chambers and injection ports with demineralized water and dry the chambers using

Kim wipes

• close the nitrogen gas supply

• disconnect the instrument by clicking on

• close the HS4800 Control Manger Software

• switch off the HS4800 hybridization station


(Parameters: Chamber type = OTHER: HS4800 chamber, Quantity of label target used = 100, Mass unit = Nano gram, time = 90, Tiny time unit = minutes, Volume = 100, Volume unit = Nano litre, temperature = 42)
P-MTAB-30122
nucleic_acid_extraction
Total RNA was isolated from Bacillus amyloliquefaciens FZB42 cultured cells using the kit NucleoSpin® RNA L (Macherey & NagelGmbH & Co.KG, Düren, Germany), according to the manufacturer's instructions. Briefly, pelleted bacterial cells were resuspended in 200 ul TE buffer (10mM Tris-HCL, 1 mM EDTA; pH 8) containing 0.2 mg/ml lysozyme by vigorous vortexing and were then incubated at 37 degrees C for 10 min. Cell lysis was proceeded by adding 1.8 ml buffer RA1 and 18ul beta-mercaptoethanol to the suspension and vortexing vigorously. Lysates were then filtered to reduce viscosity and turbidity of the solution through NucleoSpin®Filter L units, which were centrifuged for 10 min at 4,500 g. NucleoSpin® Filter L units were discarded and 1.8 ml of 70% ethanol was added to the lysates and mixed by vortexing. The lysate-ethanol mixture was subsequently loaded onto a NucleoSpin® RNA L column and centrifuged for 3 min at 4,500 g for binding of the RNA. A volume of 2.2 ml of the MDB buffer was then added to NucleoSpin® RNA L column to desalt the silica membrane, and centrifuged for 3 min at 4,500 g. DNA was removed by adding 250ul DNase reaction mixture directly onto the centre of the silica membrane and incubated at room temperature for 15 min. After three washing steps using the washing solutions RA2 and RA3, the membrane was centrifuged for 5 min at 4,500 g to dry completely and 500 ul RNase-free water was added directly onto the centre of the silica membrane, which was incubated at room temperature for 2 min and then centrifuged for 3 min at 4,500 g for elution.
(Parameters: Extracted product = total_RNA, Amplification = none)
P-MTAB-30116
labeling
Protocol for the synthesis of fluorescently labeled targets by aminoallyl coupling

(using CyScribe GFX columns for purification of aa-dUTP-labeled cDNA)

This protocol describes the contents and use of the "Bielefeld Fluorescent Labeling Kit". This kit

allows the synthesis of fluorescently labeled targets from total RNA. In a first step, aminoallyl modified

first strand cDNA can be synthesized by reverse transcription of total RNA using a mixture of

oligodT15VN and random hexamer primers.

Contents of the "Bielefeld Fluorescent Labeling Kit":

The kit contains the following reagents and solutions.

Components stored at -20 degrees C

• 5×Reaction Buffer (Bioline, delivered with BioSript Reverse Transcriptase)

• 25×dNTP stock solution (see below)

• BioScript Reverse Transcriptase (200 U/ul; Bioline)

• RNAse inhibitor (40 U/ul; Invitrogen)

• 0.2 M NaOH and 0.2 M HCl (Merck)

• 4 M hydroxylamine (Sigma, dissolve in MilliQ water)

• 1 M sodium bicarbonate pH9 (Sigma, dissolve in MilliQ water and adjust pH, pH is important)

25×dNTP stock solution:

Prepare a 25×dNTP (4:1 aa-dUTP/dTTP mix) stock as follows, store at –20 degrees C in aliquots.

• 100 mM dATP (e.g. from Peqlab): 31.25 ul (final concentration 12.5 mM)

• 100 mM dCTP (e.g. from Peqlab): 31.25 ul (final concentration 12.5 mM)

• 100 mM dGTP (e.g. from Peqlab): 31.25 ul (final concentration 12.5 mM)

• 100 mM dTTP (e.g. from Peqlab): 6.25 ul (final concentration 2.5 mM)

• 50 mM aa-dUTP (Fermentas, Life Sciences): 50.0 ul (final concentration 10.0 mM)

• H2O 100.0 ul

Components stored at 4 degrees C (-20 degrees C after aliquoting in 1/10 volumes)

• Cy3-NHS ester or Alexa555/Alexa532/Alexa546-NHS ester

• Cy5-NHS ester or Alexa647-NHS ester

• in each case, 1/6th of one aliquot of the monoreactive dye from Amersham is used for one

labeling, mg values are not stated by the supplier (estimated between 10 and 50 ?g).

• preparing aliquots: dissolve NHS esters in 10 ul of water-free DMSO, it is essential to avoid any

contact of the dyes with water prior to labeling. Immediately re-seal DMSO with fresh dessication

packs, aliquot 1.5 ul of NHS esters into 10 brown Eppendorf tubes, speed-vac in the dark for 45

min, seal dried NHS esters in plastic bags together with dessication packs, store at -20 degrees C

Components stored at RT

• CyScribe GFX columns as well as capture, washing and elution buffer (Amersham Biosciences)

You need to supply

• your favoured total RNA prepared by the methods mentioned above

• unmodified or amino-modified random hexamer primers (dissolved in DEPC-water)

• RNAse-free Eppendorf tubes and tips (e.g. from Peqlab)

• autoclaved MilliQ water

• DEPC-treated water (e.g. from the Qiagen RNeasy kit)

• 80% ethanol (diluted to 80% from absolute ethanol, Merck)

Protocol for labeling reverse-transcribed total RNA for microarray hybridizations

• time required: 4-5 hours including checking target labeling on agarose gels

• preheat the 42 degrees C and 70 degrees C heating blocks 30 min before starting and prepare an ice bucket

• wear gloves. Use filter tips, autoclavable pipetmen and RNAse-free Eppendorf tubes

• thaw DEPC H20, 5xReaction Buffer and primers

Reverse transcription of total RNA to yield aminoallyl-labeled first-strand cDNA

• mix by flicking and spin down:

• 10 to 30 ?g of total RNA purified using Microcon-30 filters up to 18.8 ul

• random hexamers [5 ?g/ul] 2.0 ul

• OR amino-modified random hexamers [5 ?g/ul] 2.0 ul

• add DEPC-treated H2O to 20.8 ul (duplication of volume is possible until the clean-up step!)

• incubate at 70 degrees C for 10 min in a heating block

• incubate at 0 degrees C for 5 min on ice (primer annealing), quickly spin down

During the 0 degrees C incubation, prepare a master mix as follows (volumes are for a 1.0× mix; prepare 1.1 ×

volumes per target) in an RNAse-free Eppendorf tube.

• 5×Reaction Buffer 6.0 ul

• RNase inhibitor [40 U/ul] 0.5 ul

• BioScript RT [200 U/ul] 1.5 ul

• 25×dNTP stock solution including aa-dUTP 1.2 ul

(4:1 aa-dUTP/dTTP nucleotide mix)

• mix by flicking, spin down, leave at RT until use. RNAse Inhibitor, BioScript and 25×dNTP should

be added immediately before use (do NOT store this mix on ice)

• at RT, add 9.2 ul of the master mix to each annealing reaction, mix by flicking, spin down

• incubate at 42 degrees C for 90 min in a heating block

• place 0.2 N NaOH, 0.2 N HCl, 1 M sodium bicarbonate and 4 M hydroxylamine at RT

from now on, RNAse-free conditions are not required

• either (for CyScribe GFX purification): wearing gloves, place CyScribe GFX columns (one per

labeling) in collection tubes and prepare one empty 1.5 ml tube per labeling. Label collection

tubes at their side. Do not label the column and make sure during the whole procedure that

columns are not mixed up. Label the 1.5 ml tubes at the side and cut off the lid.

• thaw and vortex 1 M sodium bicarbonate, pH 9.0 to dissolve white precipitates

• prepare 0.1 M sodium bicarbonate (pH 9.0) by diluting the 1 M stock solution in MilliQ water,

vortex 1 M sodium bicarbonate (pH 9.0) to dissolve white precipitates, 60 ul 0.1 M sodium

bicarbonate (pH 9.0) will be required per labeling reaction. Prepare 80% ethanol from absolute

ethanol using MilliQ water (1.8 ml per labeling).

Hydrolysis of RNA

• add 15 ul of commercial (DO NOT prepare yourself) 0.2 M NaOH (arrested pipetman!) using

100 ul filter tips (exact flowout)

• mix by flicking and spin down

• incubate at 70 degrees C for 10 min in a heating block

• add 15 ul of commercial (DO NOT prepare yourself) 0.2 M HCl using the arrested pipetman and

100 ul filter tips (exact flowout), mix immediately by pipetting up and down to avoid precipitates

• either (for CyScribe GFX purification): immediately after each target is neutralized (do not

neutralize all parallel targets first), quickly proceed with CyScribe GFX column purification

In these and the next steps, do NOT use Tris-containing buffers instead of water, since the amino

groups will interfere with the subsequent coupling

Clean-up of aminoallyl-labeled first-strand cDNA

• perform clean-up of aminoallyl-labeled first-strand cDNA using either the following Cyscribe GFX

(recommended) or the Microcon-30 protocol (more time-consuming)

Clean-up of aminoallyl-labeled first-strand cDNA (removal of nucleotides and other low-molecularweight

molecules with amino groups) using CyScribe GFX columns

• directly after neutralization of one labeling reaction add 450 ul capture buffer to the reaction and

mix by pipetting up and down (proceed with this step until all labeling reactions have been

neutralized and mixed with capture buffer), samples should not stay in capture buffer longer than

5 min

• add the complete neutralized mix to a CyScribe GFX column

• spin at 13.000 rpm for 30 sec at 20 degrees C in a microcentrifuge and discard flowthrough

• add 600 ul of 80% ethanol (it is very important not to use less concentrated ethanol, do not use

the washing buffer provided with the CyScribe GFX purification kit)

• spin at 13.000 rpm for 30 sec at 20 degrees C in a microcentrifuge and discard flowthrough

• repeat this washing step twice

• spin at 13.000 rpm for 10 sec at 20 degrees C in a microcentrifuge and place column in a new 1.5 ml tube

• add 60 ul 0.1 M sodium bicarbonate (pH 9.0), do not use the elution buffer provided with the

CyScribe GFX purification kit

• incubate for 5 min at room temperature

• spin at 13.000 rpm for 1 min at 20 degrees C in a microcentrifuge

• storage of first strand cDNA eluted from the column in 0.1 M sodium bicarbonate at -20 degrees C is not

recommended by the manufacturers of aminoallyl fluorescent labeling kits, manufacturers

recommend to proceed immediately with the coupling of fluorescent dyes (below), we have not

observed a negative effect of storage of first strand cDNA in 0.1 M sodium bicarbonate at -20 degrees C

overnight

Coupling of fluorescent dyes to the aminoallyl-labeled first-strand cDNA

• protect samples from light all the time using brown Eppendorf tubes, avoid room light and

direct sunlight

• dissolve Cy3- or Cy5-NHS or Alexa-NHS esters provided in aliquoted form (see above) in brown

Eppendorf tubes in the complete aa-containing first strand cDNA by pipetting up and down

several times until the dye is dissolved (red/blue colour!)

alternatively, 1.5 ul of fluorescent dye in DMSO (orgininal dye pack aliquot from Amersham

dissolved in 10 ul DMSO, cannot be stored, always prepare fresh!) can be added to the aacontaining

first strand cDNA solution

• from now on, work in brown Eppendorf tubes to protect the fluorescent dyes

• do NOT spin down, just tap down drops from the side of the Eppendorf tubes

• incubate for 1 h at RT in the dark (up to 2 h is possible)

Quenching (blocking of all remaining dyes with the amino groups from the hydroxylamine)

• add 4.5 ul of 4 M hydroxylamine

• mix by flicking, do NOT spin down

• leave for 15 min at RT in the dark

Clean-up of fluorescently labeled targets using CyScribe GFX Purification Kit (Amersham Biosciences)

Cy5- and Cy3-labeled targets to be hybridized simultanuously to one microarray are cleaned up

together. In the event that only one of either dye is used for hybridization or has to be purified for other

purposes, use the same volumes as specified below.

• work quickly to protect labeled targets from the light, Cy5 bleaches quickly and is

particularly sensitive to high ozone concentrations!

• add 600 ul capture buffer (CyScribe GFX Purification Kit, Amersham Biosciences) to the Cy5-

labeled sample and mix by pipetting up and down, then add the Cy3-labeled sample to this

solution and mix by pipetting up and down

• apply all to a GFX column in a collection tube (CyScribe GFX Purification Kit), do not leave the

sample in capture buffer for more than 5 min

• spin at full speed (appr. 10.000-13.000 rpm) for 30 sec

• discard flowthrough

• add 600 ul washing buffer (CyScribe GFX Purification Kit)

• spin at full speed (appr. 10.000-13.000 rpm) for 30 sec

• discard flowthrough

• add 600 ul washing buffer (CyScribe GFX Purification Kit)

• spin at full speed (appr. 10.000-13.000 rpm) for 30 sec

• discard flowthrough

• add 600 ul washing buffer (CyScribe GFX Purification Kit)

• spin at full speed (appr. 10.000-13.000 rpm) for 30 sec

• discard flowthrough

• spin at full speed (appr. 10.000-13.000 rpm) for 10 sec

• transfer the dried GFX column to a fresh brown Eppendorf tube labeled at the side, since the

cover lid has to be cut off

• add 60 ul elution buffer (CyScribe GFX Purification Kit) to the center of the filter

• leave for 5 min at RT

• spin at full speed (appr. 10.000-13.000 rpm) for 1 min

• the resulting 60 ul of combined Cy3/Cy5-labeled targets are transferred to a fresh brown tube with

a screw cap. Label on the sides and seal with "Tesafilm".

• remove 2 ul into a normal Eppendorf tube for target checking on agarose gels

• freeze this aliquot as well as the 58 ul Cy-labeled target at -20 degrees C until use
(Parameters: Amount of nucleic acid labeled = 10, Amplification = none, Mass unit = Micro gram)
P-MTAB-30118
grow
Maize seeds (Zea mays L. var. Surprise) were shaken for 3 min in 96% ethanol, 30 min in 3% sodium hypochlorite solution, rinsed twice in sterile distilled water (SDW) and then left to soak in SDW for 4 h at 25oC. Sterility of seeds was confirmed by the absence of microbial growth in liquid Luria-Bertani (LB) and semi-solid Tryptic Soy Agar media (TSA, 0.3% Agar) to which seeds had been added and incubated for seven days at 37oC. Three batches containing one hundred seeds (approximately 36 g) were added to 100 milliliters (mL) of sterile distilled water placed in 500 mL foil-wrapped Erlenmeyer flasks and kept at 28 degrees C for 12 hours on a rotary shaker at 120 rpm. After collection, 100 microliters (ul) of each sample was inoculated to LB plates to check for sterility. Contaminated batches of seeds were discarded. A single overnight colony of Bacillus amyloliquefaciens FZB42 was transferred to a culture medium containing 0.7% tryptone, 0.3% peptone, 0.1% glucose, 0.5% NaCl and 0.1% glucose (1C). When the optical density at 600 nm (OD) reached 1.0, an aliquot corresponding to 1% of the final culture volume was added to 1C medium supplemented with 10% of soil extract. Bacterial cells were incubated at 24oC, 210 rpm and then harvested at OD 3.0, which corresponded to the transient phase. Seed exudates were added into the main culture up to a final concentration of 250 µg of dry weight per mL of culture medium. Three biological replicates were obtained for control bacterial cultures as well as the treatment supplemented with seed exudates.
(Parameters: time unit = seconds, temperature unit = C)
P-MTAB-30120
bioassay_data_transformation
standard t-test
P-MTAB-30121
image_acquisition
Slides were scanned upside down on the Tecan Reloaded Scanner using the confocal mode.
(Parameters: Scanning hardware = OTHER: Tecam Reloaded, Scanning software = AIDA [Raytest GmbH])
P-MTAB-30119
pool
The experiment did not involve any pooling of samples.
P-MTAB-30114
grow
Maize seeds (Zea mays L. var. Surprise) were shaken for 3 min in 96% ethanol, 30 min in 3% sodium hypochlorite solution, rinsed twice in sterile distilled water (SDW) and then left to soak in SDW for 4 h at 25oC. Sterility of seeds was confirmed by the absence of microbial growth in liquid Luria-Bertani (LB) and semi-solid Tryptic Soy Agar media (TSA, 0.3% Agar) to which seeds had been added and incubated for seven days at 37oC. Three batches containing one hundred seeds (approximately 36 g) were added to 100 milliliters (mL) of sterile distilled water placed in 500 mL foil-wrapped Erlenmeyer flasks and kept at 28 degrees C for 12 hours on a rotary shaker at 120 rpm. After collection, 100 microliters (ul) of each sample was inoculated to LB plates to check for sterility. Contaminated batches of seeds were discarded. A single overnight colony of Bacillus amyloliquefaciens FZB42 was transferred to a culture medium containing 0.7% tryptone, 0.3% peptone, 0.1% glucose, 0.5% NaCl and 0.1% glucose (1C). When the optical density at 600 nm (OD) reached 1.0, an aliquot corresponding to 1% of the final culture volume was added to 1C medium supplemented with 10% of soil extract. Bacterial cells were incubated at 24oC, 210 rpm and then harvested at OD 1.0, which corresponded to the exponential phase. Seed exudates were added into the main culture up to a final concentration of 250 µg of dry weight per mL of culture medium. Three biological replicates were obtained for control bacterial cultures as well as the treatment supplemented with seed exudates.
(Parameters: time unit = seconds, temperature unit = C)
P-MTAB-30113
specified_biomaterial_action
The bacterial culture was mixed to a buffer composed of 20 mM Tris-HCl, 25 mM MgCl2 and 20 mM NaN3) at a ratio of 1:2, and then centrifuged at 5000 rpm, at ambient temperature, for 4 min. The supernatant was discarded and the pellet was resuspended in 1 mL of the same buffer, and centrifuged again at 9000 rpm and 4oC for 4 min. The pellets were then stored at –80 degrees C after being snap frozen in liquid nitrogen. Total RNA was isolated from 15 mL bacterial cultured cells at exponential phase (OD 1.0), following the manufacturer's instructions of the RNA purification kit NucleoSpin®RNA L (MACHEREY-NAGEL GmbH & Co.KG, Düren, Germany). The Cy3 dye was used to label the cDNA derived from the treatment and the Cy5 to label the cDNA derived from the control.
P-MTAB-30115
specified_biomaterial_action
The bacterial culture was mixed to a buffer composed of 20 mM Tris-HCl, 25 mM MgCl2 and 20 mM NaN3) at a ratio of 1:2, and then centrifuged at 5000 rpm, at ambient temperature, for 4 min. The supernatant was discarded and the pellet was resuspended in 1 mL of the same buffer, and centrifuged again at 9000 rpm and 4oC for 4 min. The pellets were then stored at –80 degrees C after being snap frozen in liquid nitrogen. Total RNA was isolated from 15 mL bacterial cultured cells at transient phase (OD 3.0), following the manufacturer's instructions of the RNA purification kit NucleoSpin®RNA L (MACHEREY-NAGEL GmbH & Co.KG, Düren, Germany). The Cy3 dye was used to label the cDNA derived from the treatment and the Cy5 to label the cDNA derived from the control.