All the slides for a same project are scanned at a constant PMT power (i.e. 650V)
5 ug of aRNA (8ul) mixed with 2 ul of random nonamers 1ug/ul and 0.5 ul of RNAse Out 40 U/ul, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample: 4 ul of first strand buffer 5 X, 1 ul of 10 mM dNTP/2mM dCTP, 2 ul of 0.1 M DTT, 1.5 ul of Cy5-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 ul of SuperScript II RT 200U/ul. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 ul of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 ul of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).
Slide preparation In a Coplin jar, prepare 50 ml of pre-hybridization solution :
SSC 20 X 12,5 ml
BSA 10 % 5 ml
SDS 10 % 0,5 ml
H2O 32 ml
Pre-heat this solution at 42 °C and préhybridize slides at 42 °C for at least 45 mn.
Wash the slides in milli-Q water, and then isopropanol.
Blow-dry the slides.
THE SLIDES MUST BE USED WITHIN 1 HOUR!
Prepare 2X hybridation mix (50 ul per slide) :
SSC 20x 25 ul
SDS 10% 1ul
Formamide 25 ul
Denature the probe 1 min at 95°C and keep on ice.
Add one volume of 2X hybridation mix, mix and centrifuge.
Put the slide in the Corning hybridization chamber, and place a cover slip on it.
Pipet the probe between the slide and the cover slip.
Put 10 ul of water in the two holes and close the chamber.
Incubate in a water bath at 42 °C over-night (15h)
Soak in 2X SSC, 0,2 % SDS at 42°C to remove the cover slip, then wash in :
- 1X SSC, 0,2 % SDS (42°C) 4 min
- 0,2X SSC, 0,2 % SDS 4 min
- 0,05X SSC 4 min
Blow dry the slide or centrifuge for 2 min at 600 rpm.
Store in a dry and dark place until scanning.
5 ug of aRNA (8ul) mixed with 2 ul of random nonamers 1ug/ul and 0.5 ul of RNAse Out 40 U/ul, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample: 4 ul of first strand buffer 5 X, 1 ul of 10 mM dNTP/2mM dCTP, 2 ul of 0.1 M DTT, 1.5 ul of Cy3-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 ul of SuperScript II RT 200U/ul. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 ul of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 ul of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).
Preparation of Lysates from Tissue Samples >30mg
This protocol describes the lysis and homogenization of tissues using 1ml of SV
RNA Lysis Buffer. Refer to Table 1 for recommended sample mass for different
1. Transfer 1ml of SV RNA Lysis Buffer (with BME added) into the tube to
which your tissue sample will be added. Use RNase-free pipettes, and wear
gloves to reduce the chance of RNase contamination.
2. Weigh the tube containing the SV RNA Lysis Buffer and record the weight.
3. Excise the tissue of interest, and place it in the tube containing the SV RNA
Lysis Buffer. Work as quickly as possible. Homogenize the tissue at high
speed using a small homogenizer (such as a Tekmar Tissuemizer) until
no visible tissue fragments remain (see Section VIII.E for additional information
on preparing lysates).
4. Weigh the tube containing the tissue and SV RNA Lysis Buffer. Calculate
the tissue mass by subtracting the weight obtained in Step 2 from this new
weight. In general, the ratio of tissue mass to SV RNA Lysis Buffer should
be approximately 171mg/ml but may be adjusted for certain tissues (see
Table 1 for recommendations for tissue mass to SV RNA Lysis Buffer ratios
for different tissues). If necessary, add SV RNA Lysis Buffer to the tissue to
achieve this ratio.
5. Transfer 175ul of the tissue lysate to a 1.5ml microcentrifuge tube.
Additional lysate should be frozen at -20C or -70C. Add 350ul of SV RNA
Dilution Buffer (blue). Mix by inverting 3-4 times. Place in a water bath or
heating block at 70C for 3 minutes. Incubation for longer than 3 minutes
may result in compromised integrity of the RNA.
6. Centrifuge for 10 minutes at 12,000-14,000 x g. Proceed to Section IV.E
(Spin Purification) or Section IV.F (Vacuum Purification).
D. Lysis of Cultured Cells
Use the following protocol for lysis of cultured cells from suspension or adherent
cultures. Use at least 1.5 x 103 cells to a maximum of 5 x 106 cells per purification.
The number of cells used may need to be adjusted depending on cell type,
function and RNA expression levels at the time of harvest.
1. For harvesting adherent cells, follow the protocol in the appendix (Section
VIII.F) prior to cell lysis. For suspension cells proceed to Step 2.
2. Collect 1.5 x 103-5 x 106 cells in a sterile 50ml conical centrifuge tube by
centrifugation at 300 x g for 5 minutes. Wash the cell pellet with 25ml of icecold,
sterile 1X PBS (see Section VIII.G for recipe). Centrifuge at 300 x g for
5 minutes to collect the cells. Discard the supernatant.
3. Verify that BME has been added to the SV RNA Lysis Buffer. Add 175ul of
SV RNA Lysis Buffer to the washed cells, dispersing the pellet and mixing
well by vortexing and/or pipetting.
4. Up to 1 x 106 cells will lyse easily in 175ul of SV RNA Lysis Buffer.
1 x 106-5 x 106 cells will need to be passed through a 20-gauge needle to
shear the genomic DNA. Repeat 4 to 5 times. Expel the lysate into a 1.5ml
5. Add 350ul of SV RNA Dilution Buffer (blue) to 175ul of lysate. Mix by
inverting the tube 3-4 times. Place in a water bath or heating block at 70C
for 3 minutes. Incubating longer than 3 minutes may compromise the
integrity of the RNA.
6. Centrifuge at 12,000-14,000 x g for 10 minutes at 20-25C. Proceed to
Section IV.E (Spin Purification) or Section IV.F (Vacuum Purification).
E. RNA Purification by Centrifugation (Spin)
Five Spin Column Assemblies (Spin Basket and 2ml Collection Tube) and five
capped Elution Tubes (1.5ml) are packed together. Wear gloves and open the
pack carefully. Remove one Spin Column Assembly for each sample to be
processed. Tape the package shut. Each Spin Column Assembly consists of a
Spin Basket and a Collection Tube. If you do not need the caps on the Spin
Baskets simply remove them using a twisting motion; the caps are designed to
detach from the Spin Baskets. If the caps are left attached to the Spin Baskets,
they must be closed during centrifugation steps. Label the Collection Tube and
place the Spin Column Assembly in a microcentrifuge tube rack. It is important
to label your tubes to maintain sample identity. Wear gloves when handling the
Note: If the lysate is too viscous to pipet easily, dilute with additional SV RNA Lysis Buffer before adding SV RNA Dilution Buffer in Step 5. Add the minimum amount of SV RNA Lysis Buffer required to make the lysate easy to pipet.
1. Transfer the cleared lysate solution to a fresh microcentrifuge tube by pipetting.
Avoid disturbing the pelleted debris.
2. Add 200ul 95% ethanol to the cleared lysate, and mix by pipetting 3-4
times. Transfer this mixture to the Spin Column Assembly. Centrifuge at
12,000-14,000 x g for one minute.
3. Take the Spin Basket from the Spin Column Assembly, and discard the
liquid in the Collection Tube. Put the Spin Basket back into the Collection
Tube. Verify that the SV RNAWash Solution has been diluted with ethanol
as described in Section IV.A. Add 600ul of SV RNAWash Solution to the
Spin Column Assembly. Centrifuge at 12,000-14,000 x g for one minute.
4. Empty the Collection Tube as before and set in a rack. For each isolation to
be performed, prepare the DNase incubation mix by combining 40ul Yellow
Core Buffer, 5ul 0.09M MnCl2 and 5ul of DNase I enzyme per sample in a
sterile tube (in this order). Prepare only the amount of DNase incubation mix
required and pipet carefully. Mix by gentle pipetting; do not vortex. Keep
the DNase I on ice while it is thawed. Apply 50ul of this freshly prepared
DNase incubation mix directly to the membrane inside the Spin Basket. It is
important to make sure that the solution is in contact with and thoroughly
covering the membrane. The incubation solution is yellow to make this
easier to visualize.
5. Incubate for 15 minutes at 20-25C. After this incubation, add 200ul of SV
DNase Stop Solution (as prepared in Section IV.A; verify that ethanol has
been added) to the Spin Basket, and centrifuge at 12,000-14,000 x g for
one minute. There is no need to empty the Collection Tube before the next
6. Add 600ul SV RNA Wash Solution (with ethanol added) and centrifuge at
12,000-14,000 x g for one minute.
7. Empty the Collection Tube, and add 250ul SV RNA Wash Solution (with
ethanol added); centrifuge at high speed for two minutes.
8. If you have not already done so, remove the cap from the Spin Basket by
using a twisting motion.
9. For each sample, remove one capped 1.5ml Elution Tube from the packaging.
Tape the package shut. Note that there is only one Elution Tube per
Spin Column Assembly. Transfer the Spin Basket from the Collection Tube
to the Elution Tube, and add 100ul Nuclease-Free Water to the membrane.
Be sure to completely cover the surface of the membrane with the water.
Place the Spin Basket Assemblies in the centrifuge with the lids of the
Elution Tubes facing out. Centrifuge at 12,000-14,000 x g for one minute.
Remove the Spin Basket and discard. Cap the Elution Tube containing the
purified RNA and store at -70C.
Media: 0.5 mM CaSO4, 5 mM KNO3, 0.5 mM MgCl2, 1 mM KH2PO4, 50 uM Na2FeEDTA, 50 uM H3Bo3, 15 uM MnCl2, 1 uM ZnCl2, 1 uM CuCl2, 0.03 uM (NH4)6Mo7O24 and 2.5 mM MES, with pH adjusted to 5.7 with KOH; plus agar 0.8 % (v/w).
hygrometry : 70%
Temperature : 20 C
Light : 180 umol m-2 s-1, 16 h per day
Media : 125 uM CaSO4, 125 uM KNO3, 125 uM MgCl2, 250 uM KH2PO4, 12.5 uM Na2FeEDTA, 12.5 uM H3Bo3, 3.75 uM MnCl2, 0.25 uM ZnCl2, 0.25 uM CuCl2, 7.5 nM (NH4)6Mo7O24 and 625 uM MES, with pH adjusted to 5.7 with 1 M KOH.
hygrometry : 70%
Temperature : 21-23 C
Light : 155 umol m-2 s-1, 8 h per day
Sample Treated names :
- control infiltrated leaves
- Bradyrhizobium infiltrated leaves
- Bradyrhizobium pathogen-infiltrated leaves
- control pathogen-infiltrated leaves
Treatment date month/day/year : 04/30/2004
Treatment name : a combination of 2 treatments have been made, namely: Bradyrhizobium, pathogen.
Treatment factor : Pathogen infection = PGPR.
Treatment Condition : In vivo.
Factor name : the PGPR strain Bradyrhizobium ORS278, and the pathogenic strain Pseudomonas syringae pv. tomato DC3000.
Factor value : 107 cfu.g-1 of soil for Bradyrhizobium and 2.105 cfu.ml-1 in MgSO4 10 mM infiltrated in leaves for Pseudomonas syringae
Time elapsed treatment : 4 weeks and 24 h for the Bradyrhizobium and pathogen treatments respectively
Protocol : Seeds were sawn on 0.8% (W/V) agar mineral medium (see below). 4 days after storage in the dark at 4C, seedling were cultivated 6 days in a growth chamber (16 h daily, 20-22C) and then transferred on soil inoculated or not with 107 cfu.g-1 of Bradyrhizobium strain ORS278. Three weeks later, 3 leaves per plant were infiltrated with a suspension of Pseudomonas syringae pv. tomato (2.105 cfu.ml-1) or with MgSO4 10 mM alone for control plants. Infiltrated leaves were collected 24h later.
Sample Treated names :
- Leaves_ Phyllobacterium
Treatment date month/day/year : 05/24/2004
Treatment name : Mesorhizobium, Phyllobacterium, Bradyrhizobium.
Treatment type : Environmental treatment.
Treatment factor : Rhizobacteria inoculation.
Treatment Condition : In vivo.
Factor name : Mesorhizobium, Phyllobacterium, Bradyrhizobium
Factor value : 108 cfu.g-1 of soil for the Mesorhizobium and Phyllobacterium treatments, 107 cfu.g-1 of soil for the Bradyrhizobium treatment.
Time elapsed treatment : 4 weeks
Protocol : Seeds of wild-type Arabidopsis thaliana (ecotype Columbia) were surface-sterilized and sawn on agar mineral medium. Four days after storage in the dark at 4C, seedlings were cultivated 6 days in a growth chamber (16 h daily, 20-22C) and then transferred on soil inoculated or not with 108 cfu.g-1 of Mesorhizobium loti, or 108 cfu.g-1 of Phyllobacterium STM196, or 107 cfu.g-1 of Bradyrhizobium ORS278.
Sample Treated names :
- roots_Phyllobacterium in vitro
- leaves Phyllobacterium in vitro
Treatment date month/day/year : 11/10/2004
Treatment name : Phyllobacterium
Treatment factor : PGPR
Factor value : 2.108 cfu Phyllobacterium per ml medium
Time elapsed treatment : 6 days
Protocol : Seeds of wild-type Arabidopsis thaliana (ecotype Columbia) were surface-sterilized and sawn on agar mineral medium (see below). 4 days after storage in the dark at 4C, seedling were cultivated 6 days in a growth chamber (16 h daily, 20-22C) and then transferred on a fresh agar mineral medium inoculated or not with Phyllobacterium STM196 (2.108 cfu/ml). 6 days later, root and leaves were collected, froze on liquid nitrogen and stored at -80C.