{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"submitter":["philip poole"],"study_type":["transcription profiling by array"],"organism":["Rhizobium leguminosarum"],"species":["Rhizobium leguminosarum"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MEXP-2901"],"description":["Rhizobium leguminosarum biovar viciae strain RU2386 was grown overnight on glc asp and the next day it was resuspended in glucose glutamate. Gene expression before resuspension was compared to the 2 h resuspended sample."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Hybridization - Hybridization using Slide Booster_18hrs Version1



Entered by KK 24 July 2008



Materials used:



-20XSSC**

-10% SDS Invitrogen Cat No 15553-027 Lot no 1239868

-Deionised Formamide Sigma F9037 (kept in fridge)

-Calf Thymus DNA Sigma D8661

-Water Sigma Cat NoW4502

-Corning Ultra gaps slides (Printed with Rhizobium oligoM-^Rs)

-Slide Booster Booster (http://www.implen.co.uk/product.php5?id=17)

-LifterSlips(Implen 25x60)

-50ml conical centrifuge tubes (Fisher/Corning)

-Plastic black Trough VWR 406023600

-Plastic Black Holders VWR 406023700

-MilliQ water sterile (ASK CSS TO MAKE)

-Air Duster Plus Electrolube Cat No EADP400

-TPP 0.22um FILTERTOP 250ml- Cat No 99255T

-DNA Mini Speed Vac (Streptomyces Laboratory)

-Humidifying Buffer Advalytix AdvaHum Cat No101 Lot 102050215002

-Coupling Liquid (Advalytix AdvaSon AS100)

-1.5ml RNase Free Micro centrifuge tubes (Eppendorf Cat No 0030120191) Lot No T1189680)

-AMBER TUBES



**20XSSC

175.3g NaCl ; 88.2g Sodium Citrate in 800ml MilliQ water.

Adjust the pH 7.0 with HCl.

Make the volume to 1L using MilliQ water. Autoclave and store at RT.



Hybridization:



-Clean the LifterSlips with Sterile MilliQ water and wash them with 70% Ethanol and rinse again with Sterile MilliQ water

-Flush with pressurized air to get rid off the dust





Before preparing the target turn on the Laptop and run the Booster Control Version 3.2.





Add 500ul (if you are using one slide) or 1000ul if you are using two slide on the little plastic trays



Add 15ul (X3) of coupling liquid (three places on the advalytix mixer chip)



Place the Sodium borohydeide blocked slide above the coupling liquid. Adjust them (the way that the slide is in contact with the advalytix mixer chip). Place the LifterSlip above the slide.



Settings Mixing Power M-^V 27



Pulse/Pause Ratio: 3:7



Time-18hrs at 42M-:C



Prepare the target (cDNA) approximately 55ul (about 25-40pmols) of the Labelled Target and Hybridization buffer for Lifterslips



Cy dyes are light sensitive so careful when you are working with it by turn off the lights before drying and handing





-Combine the Cy3 and Cy5 (25-40pmols) labelled cDNA targets together (about 50ul) in a Amber Centrifuge tube

-Vacuum Dry (usually at 37M-:C) the mixed labels using Minidry Vacuum centrifuge (The rotor has to wipe with RNAZap before use)

-Make the Hybridization Buffer (25% Deionised formamide, SSc 5X, SDS 0.1%) and store at 42?C until use

-Add 55ul of Hybridization buffer to the dried pellet

-(DonM-^Rt Dry the Pellet completely and leave about 3-5ul at the end it. It takes approximately 60-90minutes)

-Denature the target at 95?C for 2 minutes, protecting from light

-Leave at RT for 2minutes and centrifuge at 13000 rpm for 2 minutes to collect the condense

-Load 55 ul sample onto Slide without air bubbles

-Incubate overnight (18hrs) at 42?C in Slide booster



Removal of LifterSlip after the Hybridization



-Carefully remove the LifterSlip along with the slide

-Place them into 100ml of Wash Buffer-I in sterile Beaker)

-Carefully remove the Slide away from the LifterSlips

-Place them immediately onto Black trough filled with Wash Buffer-I pre-warmed at 42?C



Washing Protocol

-Wear gloves and keep the tweezers ready

-Remove the Advacard carefully from the Array Booster and place them in sterile 100ml corning beaker (from CSS) containing wash buffer 1 preheated to 42M-:C and carefully remove the slide with Advacard using tweezers and shake in orbital shaker (100rpm) for 7minutes. (Two slides per beaker)

-Transfer to 0.2M-WSSC, 0.1 % (w/v) SDS (WASH BUFFER 2 preheated to 42M-:C) in a black plastic trough, shake for 5 min in orbital shaker (100rpm). Up to two slides per trough at either end.

-Transfer to 0.2M-WSSC, 0.1 % (w/v) SDS (WASH BUFFER 2 preheated to 42M-:C) in a black plastic rack, shake for 5 min in orbital shaker (100rpm)

-Transfer to 0.2M-WSSC (RT) (WASH BUFFER 3) in a black plastic rack, shake for 4 min in orbital shaker (100rpm)

-Transfer to 0.2M-WSSC (RT) (WASH BUFFER 3) in a black plastic rack, shake for 4 min in orbital shaker (100rpm)

-Transfer to 0.1M-WSSC (WASH BUFFER 4) in a black plastic rack, and shake for 1min in orbital shaker (100rpm)

-Transfer into 50ml Falcon Tubes filled with 45ml of Sterile Milli-Q and dip 2-3 times

-Transfer into 50ml Falcon Tubes filled with 35ml of 2- Propanol and dip 2-3 times

-Transfer the slide to a slide holder. Spin the slide with the barcode at the bottom of the Falcon tube and facing upwards at 800rpm for 5 min and examine for dust particles

-If you are not satisfied dip in Milli-Q and spin down immediately using above method

-Place the slides in Corning Slide Box and scan them within 1 hour


(Parameters: Chamber type = OTHER: Implen Slide Booster, Quantity of label target used = 5, Mass unit = Micro gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 42)","Nucleic Acid Extraction - All Gilson tips should be filter barrier tips guaranteed to be RNA free (Axygen filter barrier tips Thistle Scientific). Gilsons (Currently we use a dedicated RNA set of Nichipet pipettes) should be wiped with RNA Zap before working and laid on paper towel. Benches should be scrupulously clean (wipe with Tegodyne before use) and gloves must be worn at all times, you are one of the main sources of RNAases. As a rule we do not make up our own solutions, wherever possible they should be bought in as reagent solutions guaranteed to be RNAase free. If it is unavoidable to weight out a chemical then spatulas must be dry oven baked or wiped with RNA Zap to remove RNAases before using them. Make sure the chemical is weighed into an RNAase free container (e.g. Falcon tube) and add RNAase free water (e.g. Sigma water cat no W4502). Water is made RNAase free by adding 0.1% DEPC, keep overnight at 37oC and autoclave the next day. Most containers should be disposable plasticware (such as Falcon tubes) but if equipment or glassware needs to be used then soak it in 0.1 N NaOH with 1mM EDTA before washing with DEPC treated water. Use sterile Falcon tubes to measure out large volumes and dispose of after each use. Amber Eppendorfs (Camlabs RTP/77120-A) are suspended overnight in 0.1% DEPC at 37oC and washed the next day in DEPC treated water (use gloved hands or RNA Zap treated forceps to recover tubes). Finally the Eppendorfs are dried and autoclaved. Alternatively, use Amber Eppendorfs tubes that are supplied sterile and guaranteed RNAase free (e.g. Eppendorf 0030 120 191). Prepare 10 mM Tris-Cl pH 8.0 (500 ul for each RNA preparation) from 1M Tris-Cl pH 8.0 stock (Sigma RNAase free cat no T2694) by diluting with Sigma (cat no W4502) RNAase free water. Prepare the dilution in a sterile Falcon tube or DEPC treated sterile Eppendorf. A small stock may be made in advance and kept for several weeks. Aliquot the required amount of RLT buffer (Qiagen RNeasy mini kit) (700M-^CM-]l for each 6 ml of culture) and add B-ME (Sigma) (10ul of B-ME for 1ml of RLT buffer). Do not keep for more than 2 weeks. Add the RLT+B-ME buffer (700 ul) to the Lysing matrix B (Q BIO gene) and incubate in ice. DNase I incubation mix: Add 10ul of DNase I with 70ul of Buffer RDD (Qiagen RNase-free-DNase set) for each 6 ml culture and mix gently. Isolation of RNA 1. Grow 3841 cells in AMS at 26oC in a shaker. 2. Take 24ml of RNA later in a 50ml Sorval tube (The Sorval tube must have been cleaned and autoclaved from a batch of tubes kept for RNA work. The RNA later added to this tube should inactivate any RNAases). 3. Add 12ml of 0.8 OD cells and vortex for 5 sec and incubate for 5 min at RT. 4. Harvest the cells at 10,000 rpm at 4oC for 10 min in the Sorval SS34 rotor. 5. Decant the supernatant and carefully dry the tube on a filter paper. 6. Add 500 ul of 10mM Tris-Cl pH 8.0 and resuspend. 7. Add 250 ul of resuspended cells to 700 ul of pre-cooled RLT buffer (with B-ME) in the Lysing Matrix B (i.e. you will need two ribolyser tubes per 12 ml of starting culture). 8. Open the cells in Ribolyser at speed 6.5 for 30 sec. 9. Incubate the tube in ice for 3 min. 10. Spin the tube in a microfuge at 13000 rpm, 4oC for 3 min. 11. Transfer the supernatant (600 ul to a new Eppendorf tube) taking care to avoid the resin. If any of the resin is disturbed then spin the supernatant again at 13000 rpm, 4oC for 3 min and transfer the supernatant to a new tube. 12. Add 450 ul of 95 % ethanol (neat AnalR) and mix well. A white precipitate may form but donM-!M-&t worry it is fine for the next step. 13. Add approx 700 ul to the RNeasy column and spin at 13000 rpm, RT for 30 sec and discard the flow through. 14. Apply the remaining material from step 12 to the RNeasy column and spin at 13000 rpm, RT for 30 sec and discard the flow through. 15. Add 350 ul of RW1 buffer into the column and spin at 13000 rpm, RT for 15 sec and discard the flow through. 16. Add 80 ul of DNase I incubation mix and incubate for 20 min in RT. 17. Add 350 ul of RW1 buffer into the column and spin at 13000 rpm, RT for 15sec and discard the flow through and the collection tube. 18. Transfer the column to a new collection tube and add 500 ul of RPE buffer. Spin at 13000 rpm, RT for 15 sec and discard the flow through. 19. Add another 500 ul of RPE buffer and spin at 13000 rpm, RT for 15 sec and discard the flow through. 20. Spin the column at 13000 rpm, RT for 2 min to dry the column. 21. Change to a final Eppendorf collection tube (lid will have to be cut off) and add 80-100 ul of RNase free water (Sigma cat no W4502) and incubate for 1 min in RT and spin at 13000 rpm, RT for 1min. 22. Cap Eppendorfs and freeze at -80oC.
(Parameters: Extracted product = total_RNA, Amplification = none)","Growth Protocol - All the growth experiments were done using 250ml Ehrlenmeyer flasks with 50 ml AMS and carbon (glucose 10mM) and nitrogen (ammonium chloride 10mM)and a 1% inoculum of Rhizobium leguminosarum strain 3841 was inoculated (500M-5l. Shaking was at 250 rpm at 26C for 16-24 hours. Cultures were harvested at an OD 600 of 0.4-0.6 Foe NaCL stressed cultures 100 mM NaCl was added.


(Parameters: start time = 16, stop time = 24, time unit = hours, min temperature = 26, temperature unit = C, media = ACID MINIMAL SALTS)","Labeling - Indirect Labelling of First M-^V Strand cDNA synthesis using Amersham Cyscribe Post- Labelling Kit



Materials and Reagents to be supplied by User



ALL REAGENTS ARE MADE WITH MILLI-Q WATER



 Cyscribe Post-Labelling kit with Cyscribe GFX purification kit - Amersham Biosciences Cat No RPN 5660X



 Cyscribe GFX purification kit Cat No 27-9606-02 (Optional)



 2.5M NaOH (1g/10ml Sterile Milli-Q water and filter sterilize with 0.45M-5 filter)



 2M HEPES (4.77g/10ml Sterile Milli-Q Water filter sterilize with 0.45M-5 filter)



 0.1M Na2CO3 (Sigma S7795) 0.53g/50ml Sterile Milli-Q water



 0.1M Sodium Bicarbonate pH9.0 (Sigma S6297) 4.2 g in 500ml Sterile Milli-Q water adjust to pH9.0 with 0.1M Na2CO3 (25- 30ml). Filter sterilize and dispense into aliquots and store at -20M-:C for up to 2 months



 90-100% Ethanol (AnalR)

 80% Ethanol



 4M Hydroxyl amine Hydrochloride Aldrich 15941-7 (27.8g in 100ml Sterile Milli-Q Water)



 Heating Block set at 65M-:C ,70M-:C and water bath at 42M-:C



 Microcentifuge



 DEPC treated Eppendorf and RNase free filter Tips (Procedures already in RNA Protocol)



 Spectrophotometer



 Microcuvettes (Brand UV-cuvettes) cat No Fischer SUPC CXA-205-020F



 Nitrile Gloves (Fisher SUPC- Cat No FB69265)





 Note : Prepare a 100mM aa-dUTP solution as follows: (Adapted from Anke BeckerM-^Rs Protocol)



Dissolve 1mg of aa-dUTP in 17M-5l DEPC-treated water (Sigma RNase free Water) and 0.68M-5l 1N NaOH. Measure pH with a drop on a pH filter paper and immediately adjust to pH7.0 using 0.1- 0.2M-5l of 1N NaOH. Store aa-(dUTP) solutions at M-^V20M-:C





** Composition of the mix (50X)



Components Volume Final Conc

100mM dATP 10.0M-5l 25mM

100mM dGTP 10.0M-5l 25mM

100mM dCTP 10.0M-5l 25mM

100mM dTTP 2.0M-5l 5mM

aa-dUTP 8.0M-5l 20mM



A: Primer Annealing



1. Add the following components to a 1.5ml Micro centrifuge tube on Ice. The RNA may need to be concentrated in a SpeediVac. Wipe out machine with RNAZap and set temp to 37oC. To reduce 16.5 M-5l to 11.5 will take about 5 min to reduce 22 M-5l to 11.5 M-5l will take about 15 min. Do not dry the sample. Reduce volume to slightly less than 11.5 M-5l then make up to 11.5 M-5l with DEPC water.



Total RNA (10-15M-5g)

11.5M-5l



Random Nanomers (from Cyscribe kit) 1M-5l





2. Mix gently by pipetting up and down



3. Incubate the reaction at 70M-:C for 5min



4. Cool reaction at RT for 10min to allow the primers and mRNA template to anneal



5. Pulse in microfuge to collect material.





B. Extension Reactions



6. Place the cooled annealing reaction on Ice and add the following components to it and make sure to add the enzyme last



5X Cyscript Buffer

4M-5l



0.1M DTT 2M-5l

50x aa-UTP/Nucleotide mix 0.5M-5l

Cyscript RT 1.0M-5l





7. Mix by stirring with the pipette tip (Cyscript is sensitive to mechanical disruption) and pulse in microfuge to collect material.

8. Incubate the reaction at 42M-:C in a water bath for 5hrs





C. Degradation of mRNA



9. Add 2M-5l 2.5M NaOH to each cDNA reaction

10. Mix reaction by vortexing and spin for 15sec

11. Incubate reaction at 37M-:C for 15min

12. Add 10M-5l 2M HEPES to each cDNA reaction

13. Mix reaction by vortexing and spin for 15sec

14. The cDNA reaction is ready for purification or can be stored at -20M-:C





D. Purification of cDNA with Cyscribe GFX purification kit



15. For every cDNA reaction to be purified, place one Cyscribe GFX column into a clean collection tube. Add 500M-5l of capture buffer to each Cyscribe GFX column

16. Briefly spin down the cDNA reaction and add into Capture Buffer and pipetting up and down 5 times

17. Centrifuge the column @13k for 30sec

18. Discard the flow through

19. Add 600M-5l of 80% Ethanol to each column and centrifuge the column @13k for 30second

20. Repeat step19 another two times

21. Centrifuge the column @13k for 10sec to remove the residual wash buffer. Discard the collection tube

22. Transfer the column into a new Amber Eppendorf tube (cat 0030 120 191) and add 60M-5l 0.1M Sodium Bicarbonate pH9.0 to the top of the glass fibre matrix

23. Incubate at RT for 1-5mins

24. Centrifuge the column @13k for 1min to collect the purified cDNA. If you leave the Eppendorf lids on then use the Heraeus Biofuge 13 with the lids turned sideways.





E. Labelling of Amino-allyl modified cDNA with cy DYES



 Turn the lights off for all subsequent steps



25. Add (60M-5l) Amino-allyl modified cDNA (0.1M Sodium Bicarbonate pH9.0) directly into an aliquot of cyDye NHS ester (from the Cyscribe kit) and Resuspend NHS ester by pipetting several times

26. Spin down @13k for 1min to collect at the Bottom

27. Incubate in Dark overnight (preferentially in the cupboard)

28. Next day add 15M-5l 4M Hydroxylamine to each coupling reaction

29. Mix by pipetting up and down and INCUBATE IN DARK at RT for 15mins



F. Purification of cyDye M-^V Labelled DNA with Cyscribe GFX purification kit



 Keep the Elution Buffer at 65M-:C



30. For every cDNA reaction to be purified, place one Cyscribe GFX column into a clean collection tube. Add 500M-5l of capture buffer to each Cyscribe GFX column

31. Microfuge pulse the cDNA reaction (from step 30) and add into Capture Buffer and pipetting up and down 5 times (Work quickly, capture buffer will degrade cy dyes)

32. Centrifuge the column @13k for 30sec (On the membrane Cy3 will look pink and cy5 blue)

33. Discard the flow through

34. Add 600M-5l of Wash Buffer to each column and centrifuge the column @13k for 30second

35. Repeat the step 35 another two times

36. Centrifuge the column @13k for 10sec to remove the residual wash buffer. Discard the collection tube

37. Transfer the column into a new Eppendorf tube and add 70M-5l Elution buffer (65M-:C) to the top of the glass fibre matrix

38. Incubate at RT for 1-5mins

39. Centrifuge the column @13k for 1min to collect the purified cDNA





Measure the absorbance of the labelled cDNA (all 70M-5l) on a spectrophotometer using the following formula:



pmole cy3 = A550/ 150 x volume(l) x 103



pmole cy5 = A650/ 250 x volume(l) x 103



pmole cDNA= A260 x volume(l) x 37ng/ml x 1000pg/ng/



324.5pg/pmole



Calculate the Nucleotides/Dye ratio and required volume for x pmole of both Cy5 and Cy3 labelled cDNA



Nucleotides/ Dye ratio = pmole cDNA/ pmole Cy Dye


(Parameters: Amount of nucleic acid labeled = 15, Label used = Cy3, Amplification = none, Mass unit = Micro gram)"],"figure_sub":["MIAME Score","Raw Data","Organization","Assays and Data","Processed Data","Additional Files","MAGE-TAB Files","Array Designs"],"pubmed_authors":["philip poole"],"data_protocol":["Assay Data Transformation - Per Spot and Per Chip: Intensity dependent (Lowess) normalization was performed within the GeneSpring version 7.3 programme.



(Also called non-linear normalization) This normalization method is used to eliminate dye-related artifacts in two-color experiments that cause the Cy5/Cy3 ratio to be affected by the total intensity of the spot. Attempts to correct for artifacts caused by non-linear rates of dye incorporation as well as inconsistencies in the relative fluorescence intensity between some red and green dyes. In the absence of bias, one would expect the raw signal to be independent of the control signal and thus the data points would be scattered symmetrically around the 45o line.

These artifacts can cause the graph of raw versus control signal to be curved. This normalization step fits a curve through the data and uses this curve to adjust the control value for each measurement. When the resulting normalized data are graphed versus the adjusted control value, the points are distributed more symmetrically around the 45o line. The percent of data to be used for smoothing can be changed from the default value of 20.0%. The raw and control values for each spot are shifted by a constant to counter the problem of taking logarithms of negative values in subsequent steps, and to discount outliers.

The process uses region designators in the same way that other per-chip normalization methods do. For use in most two-color experiments. Can be applied only to chips with more than 100 genes. A cutoff value is used if the control channel is very low. The default cutoff value is 10, but a different value can be entered in the Cutoff text box. Can be applied to all samples or selected samples only.

","Image Adquisition - Scan the slides using Axon scanner at ex Coff (80%) and variable PMT gain. Start with PMT gain at 550 and 30% power for the prescan. Do final scans at 70 % power using appropriate gain to balance the channels.
(Parameters: Scanning hardware = Axon- GenePix4000B, Scanning software = BlueFuse [BlueGnome])"],"additional_accession":[]},"is_claimable":false,"name":"Transcription profiling of Rhizobium leguminosarum biovar viciae strain RU2386 grown overnight in glucose aspartate minimial medium and next day resuspended in glucose glutamate","description":"Rhizobium leguminosarum biovar viciae strain RU2386 was grown overnight on glc asp and the next day it was resuspended in glucose glutamate. Gene expression before resuspension was compared to the 2 h resuspended sample.","dates":{"release":"2010-10-03T00:00:00Z","modification":"2022-11-21T11:42:56.556Z","creation":"2022-01-31T15:36:20.88Z"},"accession":"E-MEXP-2901","cross_references":{"EFO":["EFO_0002768"]}}