Isolation of RNA General comments Both Biophenol and Acidic phenol (Sigma) are suitable for application in the Extraction Mixture. The ratio of pellet to Extraction Mixture seems to affect the RNA amounts and quality. 50 ml MRS culture of an OD600 of 1 gives reproducibly good results. Five times this amount, however, yielded poor quality RNA (Personal communication from Arno Wegkamp and Mariela Hebben). The quenching buffer contains methanol. Supernatant from the quenching procedure should be discarded in the appropriate container. The extraction buffer contains phenol. Phenol is a toxic compound and waste containing phenol should be discarded in the appropriate containers. Collecting cells Quenching Buffer 66.7 mM HEPES (pH 6.5) 60% Methanol Extraction Mixture 500 µl Phenol/Chloroform 30 µl 10% SDS 30 µl 3 M NaAc (pH 5.2) 500 mg glass-beads (75-150 µm) 400 µl TE buffer (or MRS) Prepare in a screw-cap tube 1. For each sample prepare a tube with Extraction Mixture Make sure these components are mixed freshly each time, so do not freeze tubes containing Extraction Mixture. 2. Harvest cells and quench them immediately in Quenching Buffer. Add 4 volumes of Quenching Buffer ( 40°C) to 1 volume of cell culture 3. Mix and store the sample at -40°C 4. Centrifuge cells at 20 C at 9000 rpm for 10 minutes (Centrifuge RC5B, room A156) 5. Discard the supernatant; keep the cell pellet cooled in ethylene glycol (60 %) and work quickly to prevent condensation of water. Transfer the pellet with a pre-cooled spatula to a screw-crap tube with Extraction Mixture The supernatant contains methanol and should be discarded in the appropriate container 6. Mix the cell pellet thoroughly with Extraction Mixture by shaking by hand 7. Freeze the tube immediately in liquid nitrogen 8. Store at 80°C or isolate RNA immediately RNA isolation Tips: Work as quickly as possible and keep the samples cool. Do not break too much samples at once (max 4) to enable you to work quickly. Break the cells immediately in frozen condition. Do not thaw the samples from 80°C. Keep your samples cool during processing. Use filter tips to avoid contamination from pipets. Wear gloves and lab coat. For more tips look at the Ambion website: http://www.ambion.com The following steps should be performed as swiftly as possible before cDNA labelling. 1. Break cells in the Savant FastPrep FP120. Three times 40 seconds at speed 4.0 (cool cells between steps if necessary) 2. Centrifuge for 1 minute at 4°C in an Eppendorf centrifuge at 20.800 g. 3. Transfer 500 µl of the supernatant to a new tube. Add 400 µl chloroform (chloroform should be cooled at 4°C) 4. Centrifuge for 1 minute in an Eppendorf centrifuge at full speed (4°C). 5. Take the supernatant and mix it in 1:1 ratio with the capture buffer of the RNA-isolation kit. 6. Continue with the "High Pure RNA Isolation Kit" from Roche (order # 1 828 665) 7. Incubate for 60 minutes with DnaseI, elute with 50 µl elution buffer 8. Store the RNA in at least 2 aliquots, one of 20 µl (for concentration and quality analysis and labelling) and one back-up of 30 µl at 80°C. RNA quality control Measuring the RNA concentration Measure the concentration of RNA at 260 nm by dissolving 1 µl of a preparation in 500 µl of water. 1 absorption unit at 260 nm is equivalent to 40 µg RNA/ml. The A260/A280 ratio should be in the range of 1.9-2.0. The closer to 2.0, the more pure the RNA will be. When the ratio is lower than 1.9, DNA is still present. When lower than 1.8, proteins are still present. The recommended RNA concentration for labelling is 20-25 µg /µl . The minimal RNA concentration should be 1 µg/µl. Quality check on the Bioanalyzer First determine the RNA-concentration using the "nanodrop". The Bioanalyzer can be found in room A230. For the quality check you have to use the Nano-assay chips. Try to apply 12 samples to the Bioanalyzer, but, on the other hand, do not wait for a week to analyse your samples. Please follow the instructions of the manufacturer or ask the instrument custodian (= Anne Wiersma). The ratio between the 23S and the 16S RNA should be at least 1.6. Lower ratios are an indication of extensive RNA degradation. The "zero-line" in the electrogram should be more or less straight (when in doubt, ask an experienced researcher). The analysis on the Bioanalyzer is a qualitative assay, not a quantitative assay. The concentrations found in the spectrophotometer are more reliable.
Label
Cy3
Label protocol
General comments Tips Be sure that your RNA is of good quality before you start. Use PCR machine 3 or 4 (suitable for 0.2 ml tubes) for incubation steps, (program 70marc and 42marc). Place all the reagents (except for the RT enzyme) on ice to thaw. Chemicals descriptions can be found on page 6. All reagents are in room A128. If you take a new batch, please inform beheerder (= Marc Stevens). This protocol is made for the so-called "Home-made kit". The protocol for the Amersham post-labelling kit is the same, except for the chemical's name. Please follow the manufacturer's protocol when in doubt. A micro-array file can be found in room A128 with more information, order numbers and manufacturer's protocols. While purifying the labelled cDNA, the column should still be coloured even after tree wash steps, when you have labelled total RNA from bacteria. If you do not see the dye colours at this stage, you will probably not have enough labelled cDNA to obtain good hybridization results. Labelled cDNA can be stored at -20°C, but only for a short period. In particular the Cy5 label quickly bleaches. Do not use labelled DNA older than a few days, or you may get very low signal on the array. Cy-dye bleaching has been shown to be promoted by light, ozone and humidity. On hot summer days, or in the vicinity of UV lamps dyes may bleach very quickly. Prepared Cy-dyes can be stored up to tree months at -80°C. Note that the relation between the amount of RNA that goes into the reaction and the amount of cDNA synthesized is not proportional (see figure below by Roger Bongers) Things that can go wrong (and already went wrong once) Wash with EtOH (80%), elute with NaCO3 during the first purification. Work in the dark from the moment you see colours in your sample. Add 40 ml of 100% EtOH to the wash buffer of the GFX column purification kit. cDNA Synthesis General comments Nuclease-free water is available in PCR kits or can be ordered from commercial suppliers. It is not useful to add more than 25 µg RNA to the Annealing Mix, because this will not lead to higher cDNA yields (see figure on page 1). Annealing Annealing Mix x µl RNA preparation (25 µg RNA for spotted arrays or 5 to 10 µg RNA for Agilent arrays, see tips) y µl nuclease-free water 1 µl Random Nonamers 11 µl TOTAL 1. Mix Annealing Mix in a 200 µl PCR tube gently by pipetting up and down 2. Incubate for 5 min. 70°C 3. Cool at room temperature for 10 min. (annealing) 4. Spin down the mixture to the bottom of the tube 5. Place on ice Reverse transcription Reverse Transcription Mix 4 l 5x buffer 2 l 0.1 M DTT 2 l dNTP-mix (with AA-dUTP [home labelling kit] or 1 + 1 l for Amersham kit, see Manufacturers Protocol) 1 l TMIII (keep only briefly outside 20°C) 20 l TOTAL 1. Mix by very gently by pipetting up and down (vigorous pipetting will denature the enzyme!) Before pipetting the enzyme: spin down the residue in the tube to be sure that all enzyme is finished before starting a new kit! 2. Incubate for 3 hrs at 42°C (in PCR-machine) 3. Cool on ice for immediate purification or place at 20°C for storage Degradation of mRNA 1. Add 2 l 2,5 M NaOH 2. Mix (vortex) and spin down 3. Incubate for 15 min. at 37°C 4. Add 10 l 2 M HEPES free acid 5. Mix (vortex) and spin down 6. Ready for purification or store at 20°C Labelling of cDNA with cyanine dyes General comments Use 80% ethanol as wash buffer. The small amount of Tris present in the kit wash buffer can react with the Cy-dye NHS Esters, reducing the yield of labelling during the coupling step. Prepare fresh Sodium Bicarbonate 0.1 M, pH 9.0 (see page 6) immediately prior to use in a labelling reaction, or use an aliquot from a 20°C stock (do not freeze it again!). Purification of amino allyl-modified cDNA 1. For every cDNA reaction to be purified, place one CyScribe GFX column into a clean collection tube. Add 500 l of capture buffer to each CyScribe GFX column 2. Transfer the unpurified cDNA products into each CyScribe GFX column; mix the cDNA by gently pipetting up and down 5 times 3. Centrifuge each column in a micro centrifuge at 13.800 x g for 30 seconds 4. Remove the CyScibe GFX column and discard the liquid at the bottom of each collection tube. Return each CyScribe GFX column into the used collection tube 5. Add 600 l of 80% ethanol to each column and centrifuge at 13.800 x g for 30 seconds 6. Remove the CyScibe GFX column and discard the liquid at the bottom of each collection tube. Repeat the wash step twice for a total of three washes. After the final wash, discard the liquid and place the column back in the used collection tube 7. Centrifuge each column in a microcentrifuge at 13.800 x g for an additional 10 seconds to remove all wash buffer in the tip of the column. Discard the collection tube 8. Transfer each CyScribe GFX column to a fresh 1.5 ml micro centrifuge tube and add 60 µl of fresh 0.1 M sodium bicarbonate pH9.0 directly to the top of glass fiber matrix in each CyScribe GFX column. It is crucial that the elution buffer completely covers the membrane 9. Incubate the CyScribe GFX column at room temperature for 1-5 minutes. Centrifuge at 13.800 x g for 1 minute to collect the purified labelled cDNA 10. Repeat this elution step one more time 11. Proceed immediately to the labelling reaction Labelling of amino allyl-modified cDNA with Cy-dye 1. Add the amino allyl modified cDNA (in 0.1 M sodium bicarbonate) directly into one aliquot of CyDye NHS Ester. Resuspend the ester completely by pipetting several times and transfer the solution to an amber 1.5 ml tube 2. Incubate at room temperature, in the dark for 60 to 90 minutes 3. Add 15 µl 4 M Hydroxylamine to each coupling reaction 4. Mix by stirring and incubate at room temperature, in the dark, for 15 minutes 5. Proceed directly to purification of Cy-dye labelled cDNA Purification of Cy-dye labelled cDNA 1. For every cDNA reaction to be purified, place one CyScribe GFX column into a clean collection tube. Add 500 µl of capture buffer to each CyScribe GFX column 2. Transfer the unpurified cDNA products into each CyScribe GFX column; mix the cDNA by gently pipetting up and down 5 times 3. Centrifuge each column in a micro centrifuge at 13.800 x g for 30 seconds 4. Remove the CyScibe GFX column and discard the liquid at the bottom of each collection tube. Return each CyScribe GFX column into the used collection tube 5. Add 600 µl of wash buffer (from the kit) to each column and centrifuge at 13.800 x g for 30 seconds 6. Remove the CyScibe GFX column and discard the liquid at the bottom of each collection tube. Repeat the wash step twice for a total of three washes. After the final wash, discard the liquid and place the column back in the used collection tube 7. Centrifuge each column in a microcentrifuge at 13.800 x g for an additional 10 seconds to remove all wash buffer in the tip of the column. Discard the collection tube 8. Transfer each CyScribe GFX column to a fresh 1.5 ml amber micro centrifuge tube and add 60 µl of elution buffer (from the kit) directly to the top of the glass fiber matrix in each CyScribe GFX column. It is crucial that the elution buffer completely covers the membrane CyDye-labelled probes bind tightly to GFX resin. Never use water to elute these labelled probes because probe yield will be inconsistent and lower than that obtained with elution buffer Pre-warming elution buffer to 65°C might increase the labelled cDNA yield by approximately 5% 9. Incubate the CyScribe GFX column at room temperature for 1-5 minutes. Centrifuge at 13.800 x g for 1 minute to collect the purified labelled cDNA 10. Repeat this elution step one more time 11. Measure the concentration of cDNA, and the amount of incorporated Cy-dye using the "microarray" program of the nanodrop (see note below). 12. Ready for hybridization or store at 20°C Notes about labelled cDNA absorption spectrum The 260/280 nm absorption ratio should be in the range 1.6 to 1.8 The spectrum of the incorporated Cy-dye as recorded with the nanodrop gives you important information about the fluorescence processes going on. In particular, a high ratio of the first absorption peak relative to the second will indicate high self-quenching due to overloading of the cDNA with dye. If the peaks are almost equally high then the labelling density is too high and fluorescence will be low due to self-quenching. Home-made labelling kit components TMIII = Superscript TMIII (Invitrogen, order # 18080-044) DTT is supplied with TMIII enzyme 5 x Buffer is supplied with TMIII enzyme GFX Columns can be ordered from Amersham Bioscience, order # 27-9606-02 NaOH, HEPES, Hydroxylamine, and Sodium Bicarbonate solutions should be made by the researcher. dNTP's can be ordered from Amersham Bioscience, order # 27-2035-01 The random nonamer stock (to be diluted 1/20) can be found in room A128 Hydroxylamine 4 M Hydroxylamine Prepare freshly Sodium Bicarbonate 0.1 M, pH 9.0 0.84 g NaHCO3 90 ml Milli-Q water Adjust to exactly pH 9.0 with 1 M NaOH Adjust volume to 100 ml with Milli-Q water Store in aliquots or prepare freshly. Do not freeze again after use Random Nonamers 2 µl random nonamer stock (27.1 µg/µl) 38 µl nuclease-free water AA-dUTP 1 mg AA-dUTP (Sigma, order # A0410) 20 µl K-phosphate, 0.1M, pH 7.5 dNTP-mix 5 µl 100 mM of each dATP, dCTP and dGTP 3 µl 100 mM dTTP 2 µl AA-dUTP-mix Add 40 µl Water Cy5-dye or Cy3-dye NHS Ester 1 tube Cy5 or Cy3 monofunctional reactive dye (Amersham, order # PA23001) 40 µl DMSO Store in 8 x 5 µl aliquots at 80°C up to 3 months Use 5 µl per labelling Toxic chemicals HYDROXYLAMINE (See Chemical safety cards on http://kluyver/intranet/) Chemical dangers: May explode on heating above 70°C or when exposed to open flame. The substance decomposes rapidly at room temperature, especially in presence of moisture and carbon dioxide, and violently on heating producing toxic fumes including nitrogen oxides. The solution in water is a weak base. Reacts violently with oxidants, metals such as finely divided zinc, some metal oxides, copper(II)sulfate and phosphorus chlorides causing fire and explosion hazard. Effects of short-term exposure: The substance irritates the skin and the respiratory tract, and is corrosive to the eyes. The substance may cause effects on the blood, resulting in formation of methaemoglobin. The effects may be delayed. Medical observation is indicated. Effects of long-term or repeated exposure: Repeated or prolonged contact may cause skin sensitization. The substance may have effects on the blood, resulting in formation of methaemoglobin and the consequent anaemia.
Isolation of RNA General comments Both Biophenol and Acidic phenol (Sigma) are suitable for application in the Extraction Mixture. The ratio of pellet to Extraction Mixture seems to affect the RNA amounts and quality. 50 ml MRS culture of an OD600 of 1 gives reproducibly good results. Five times this amount, however, yielded poor quality RNA (Personal communication from Arno Wegkamp and Mariela Hebben). The quenching buffer contains methanol. Supernatant from the quenching procedure should be discarded in the appropriate container. The extraction buffer contains phenol. Phenol is a toxic compound and waste containing phenol should be discarded in the appropriate containers. Collecting cells Quenching Buffer 66.7 mM HEPES (pH 6.5) 60% Methanol Extraction Mixture 500 µl Phenol/Chloroform 30 µl 10% SDS 30 µl 3 M NaAc (pH 5.2) 500 mg glass-beads (75-150 µm) 400 µl TE buffer (or MRS) Prepare in a screw-cap tube 1. For each sample prepare a tube with Extraction Mixture Make sure these components are mixed freshly each time, so do not freeze tubes containing Extraction Mixture. 2. Harvest cells and quench them immediately in Quenching Buffer. Add 4 volumes of Quenching Buffer ( 40°C) to 1 volume of cell culture 3. Mix and store the sample at -40°C 4. Centrifuge cells at 20 C at 9000 rpm for 10 minutes (Centrifuge RC5B, room A156) 5. Discard the supernatant; keep the cell pellet cooled in ethylene glycol (60 %) and work quickly to prevent condensation of water. Transfer the pellet with a pre-cooled spatula to a screw-crap tube with Extraction Mixture The supernatant contains methanol and should be discarded in the appropriate container 6. Mix the cell pellet thoroughly with Extraction Mixture by shaking by hand 7. Freeze the tube immediately in liquid nitrogen 8. Store at 80°C or isolate RNA immediately RNA isolation Tips: Work as quickly as possible and keep the samples cool. Do not break too much samples at once (max 4) to enable you to work quickly. Break the cells immediately in frozen condition. Do not thaw the samples from 80°C. Keep your samples cool during processing. Use filter tips to avoid contamination from pipets. Wear gloves and lab coat. For more tips look at the Ambion website: http://www.ambion.com The following steps should be performed as swiftly as possible before cDNA labelling. 1. Break cells in the Savant FastPrep FP120. Three times 40 seconds at speed 4.0 (cool cells between steps if necessary) 2. Centrifuge for 1 minute at 4°C in an Eppendorf centrifuge at 20.800 g. 3. Transfer 500 µl of the supernatant to a new tube. Add 400 µl chloroform (chloroform should be cooled at 4°C) 4. Centrifuge for 1 minute in an Eppendorf centrifuge at full speed (4°C). 5. Take the supernatant and mix it in 1:1 ratio with the capture buffer of the RNA-isolation kit. 6. Continue with the "High Pure RNA Isolation Kit" from Roche (order # 1 828 665) 7. Incubate for 60 minutes with DnaseI, elute with 50 µl elution buffer 8. Store the RNA in at least 2 aliquots, one of 20 µl (for concentration and quality analysis and labelling) and one back-up of 30 µl at 80°C. RNA quality control Measuring the RNA concentration Measure the concentration of RNA at 260 nm by dissolving 1 µl of a preparation in 500 µl of water. 1 absorption unit at 260 nm is equivalent to 40 µg RNA/ml. The A260/A280 ratio should be in the range of 1.9-2.0. The closer to 2.0, the more pure the RNA will be. When the ratio is lower than 1.9, DNA is still present. When lower than 1.8, proteins are still present. The recommended RNA concentration for labelling is 20-25 µg /µl . The minimal RNA concentration should be 1 µg/µl. Quality check on the Bioanalyzer First determine the RNA-concentration using the "nanodrop". The Bioanalyzer can be found in room A230. For the quality check you have to use the Nano-assay chips. Try to apply 12 samples to the Bioanalyzer, but, on the other hand, do not wait for a week to analyse your samples. Please follow the instructions of the manufacturer or ask the instrument custodian (= Anne Wiersma). The ratio between the 23S and the 16S RNA should be at least 1.6. Lower ratios are an indication of extensive RNA degradation. The "zero-line" in the electrogram should be more or less straight (when in doubt, ask an experienced researcher). The analysis on the Bioanalyzer is a qualitative assay, not a quantitative assay. The concentrations found in the spectrophotometer are more reliable.
Label
Cy5
Label protocol
General comments Tips Be sure that your RNA is of good quality before you start. Use PCR machine 3 or 4 (suitable for 0.2 ml tubes) for incubation steps, (program 70marc and 42marc). Place all the reagents (except for the RT enzyme) on ice to thaw. Chemicals descriptions can be found on page 6. All reagents are in room A128. If you take a new batch, please inform beheerder (= Marc Stevens). This protocol is made for the so-called "Home-made kit". The protocol for the Amersham post-labelling kit is the same, except for the chemical's name. Please follow the manufacturer's protocol when in doubt. A micro-array file can be found in room A128 with more information, order numbers and manufacturer's protocols. While purifying the labelled cDNA, the column should still be coloured even after tree wash steps, when you have labelled total RNA from bacteria. If you do not see the dye colours at this stage, you will probably not have enough labelled cDNA to obtain good hybridization results. Labelled cDNA can be stored at -20°C, but only for a short period. In particular the Cy5 label quickly bleaches. Do not use labelled DNA older than a few days, or you may get very low signal on the array. Cy-dye bleaching has been shown to be promoted by light, ozone and humidity. On hot summer days, or in the vicinity of UV lamps dyes may bleach very quickly. Prepared Cy-dyes can be stored up to tree months at -80°C. Note that the relation between the amount of RNA that goes into the reaction and the amount of cDNA synthesized is not proportional (see figure below by Roger Bongers) Things that can go wrong (and already went wrong once) Wash with EtOH (80%), elute with NaCO3 during the first purification. Work in the dark from the moment you see colours in your sample. Add 40 ml of 100% EtOH to the wash buffer of the GFX column purification kit. cDNA Synthesis General comments Nuclease-free water is available in PCR kits or can be ordered from commercial suppliers. It is not useful to add more than 25 µg RNA to the Annealing Mix, because this will not lead to higher cDNA yields (see figure on page 1). Annealing Annealing Mix x µl RNA preparation (25 µg RNA for spotted arrays or 5 to 10 µg RNA for Agilent arrays, see tips) y µl nuclease-free water 1 µl Random Nonamers 11 µl TOTAL 1. Mix Annealing Mix in a 200 µl PCR tube gently by pipetting up and down 2. Incubate for 5 min. 70°C 3. Cool at room temperature for 10 min. (annealing) 4. Spin down the mixture to the bottom of the tube 5. Place on ice Reverse transcription Reverse Transcription Mix 4 l 5x buffer 2 l 0.1 M DTT 2 l dNTP-mix (with AA-dUTP [home labelling kit] or 1 + 1 l for Amersham kit, see Manufacturers Protocol) 1 l TMIII (keep only briefly outside 20°C) 20 l TOTAL 1. Mix by very gently by pipetting up and down (vigorous pipetting will denature the enzyme!) Before pipetting the enzyme: spin down the residue in the tube to be sure that all enzyme is finished before starting a new kit! 2. Incubate for 3 hrs at 42°C (in PCR-machine) 3. Cool on ice for immediate purification or place at 20°C for storage Degradation of mRNA 1. Add 2 l 2,5 M NaOH 2. Mix (vortex) and spin down 3. Incubate for 15 min. at 37°C 4. Add 10 l 2 M HEPES free acid 5. Mix (vortex) and spin down 6. Ready for purification or store at 20°C Labelling of cDNA with cyanine dyes General comments Use 80% ethanol as wash buffer. The small amount of Tris present in the kit wash buffer can react with the Cy-dye NHS Esters, reducing the yield of labelling during the coupling step. Prepare fresh Sodium Bicarbonate 0.1 M, pH 9.0 (see page 6) immediately prior to use in a labelling reaction, or use an aliquot from a 20°C stock (do not freeze it again!). Purification of amino allyl-modified cDNA 1. For every cDNA reaction to be purified, place one CyScribe GFX column into a clean collection tube. Add 500 l of capture buffer to each CyScribe GFX column 2. Transfer the unpurified cDNA products into each CyScribe GFX column; mix the cDNA by gently pipetting up and down 5 times 3. Centrifuge each column in a micro centrifuge at 13.800 x g for 30 seconds 4. Remove the CyScibe GFX column and discard the liquid at the bottom of each collection tube. Return each CyScribe GFX column into the used collection tube 5. Add 600 l of 80% ethanol to each column and centrifuge at 13.800 x g for 30 seconds 6. Remove the CyScibe GFX column and discard the liquid at the bottom of each collection tube. Repeat the wash step twice for a total of three washes. After the final wash, discard the liquid and place the column back in the used collection tube 7. Centrifuge each column in a microcentrifuge at 13.800 x g for an additional 10 seconds to remove all wash buffer in the tip of the column. Discard the collection tube 8. Transfer each CyScribe GFX column to a fresh 1.5 ml micro centrifuge tube and add 60 µl of fresh 0.1 M sodium bicarbonate pH9.0 directly to the top of glass fiber matrix in each CyScribe GFX column. It is crucial that the elution buffer completely covers the membrane 9. Incubate the CyScribe GFX column at room temperature for 1-5 minutes. Centrifuge at 13.800 x g for 1 minute to collect the purified labelled cDNA 10. Repeat this elution step one more time 11. Proceed immediately to the labelling reaction Labelling of amino allyl-modified cDNA with Cy-dye 1. Add the amino allyl modified cDNA (in 0.1 M sodium bicarbonate) directly into one aliquot of CyDye NHS Ester. Resuspend the ester completely by pipetting several times and transfer the solution to an amber 1.5 ml tube 2. Incubate at room temperature, in the dark for 60 to 90 minutes 3. Add 15 µl 4 M Hydroxylamine to each coupling reaction 4. Mix by stirring and incubate at room temperature, in the dark, for 15 minutes 5. Proceed directly to purification of Cy-dye labelled cDNA Purification of Cy-dye labelled cDNA 1. For every cDNA reaction to be purified, place one CyScribe GFX column into a clean collection tube. Add 500 µl of capture buffer to each CyScribe GFX column 2. Transfer the unpurified cDNA products into each CyScribe GFX column; mix the cDNA by gently pipetting up and down 5 times 3. Centrifuge each column in a micro centrifuge at 13.800 x g for 30 seconds 4. Remove the CyScibe GFX column and discard the liquid at the bottom of each collection tube. Return each CyScribe GFX column into the used collection tube 5. Add 600 µl of wash buffer (from the kit) to each column and centrifuge at 13.800 x g for 30 seconds 6. Remove the CyScibe GFX column and discard the liquid at the bottom of each collection tube. Repeat the wash step twice for a total of three washes. After the final wash, discard the liquid and place the column back in the used collection tube 7. Centrifuge each column in a microcentrifuge at 13.800 x g for an additional 10 seconds to remove all wash buffer in the tip of the column. Discard the collection tube 8. Transfer each CyScribe GFX column to a fresh 1.5 ml amber micro centrifuge tube and add 60 µl of elution buffer (from the kit) directly to the top of the glass fiber matrix in each CyScribe GFX column. It is crucial that the elution buffer completely covers the membrane CyDye-labelled probes bind tightly to GFX resin. Never use water to elute these labelled probes because probe yield will be inconsistent and lower than that obtained with elution buffer Pre-warming elution buffer to 65°C might increase the labelled cDNA yield by approximately 5% 9. Incubate the CyScribe GFX column at room temperature for 1-5 minutes. Centrifuge at 13.800 x g for 1 minute to collect the purified labelled cDNA 10. Repeat this elution step one more time 11. Measure the concentration of cDNA, and the amount of incorporated Cy-dye using the "microarray" program of the nanodrop (see note below). 12. Ready for hybridization or store at 20°C Notes about labelled cDNA absorption spectrum The 260/280 nm absorption ratio should be in the range 1.6 to 1.8 The spectrum of the incorporated Cy-dye as recorded with the nanodrop gives you important information about the fluorescence processes going on. In particular, a high ratio of the first absorption peak relative to the second will indicate high self-quenching due to overloading of the cDNA with dye. If the peaks are almost equally high then the labelling density is too high and fluorescence will be low due to self-quenching. Home-made labelling kit components TMIII = Superscript TMIII (Invitrogen, order # 18080-044) DTT is supplied with TMIII enzyme 5 x Buffer is supplied with TMIII enzyme GFX Columns can be ordered from Amersham Bioscience, order # 27-9606-02 NaOH, HEPES, Hydroxylamine, and Sodium Bicarbonate solutions should be made by the researcher. dNTP's can be ordered from Amersham Bioscience, order # 27-2035-01 The random nonamer stock (to be diluted 1/20) can be found in room A128 Hydroxylamine 4 M Hydroxylamine Prepare freshly Sodium Bicarbonate 0.1 M, pH 9.0 0.84 g NaHCO3 90 ml Milli-Q water Adjust to exactly pH 9.0 with 1 M NaOH Adjust volume to 100 ml with Milli-Q water Store in aliquots or prepare freshly. Do not freeze again after use Random Nonamers 2 µl random nonamer stock (27.1 µg/µl) 38 µl nuclease-free water AA-dUTP 1 mg AA-dUTP (Sigma, order # A0410) 20 µl K-phosphate, 0.1M, pH 7.5 dNTP-mix 5 µl 100 mM of each dATP, dCTP and dGTP 3 µl 100 mM dTTP 2 µl AA-dUTP-mix Add 40 µl Water Cy5-dye or Cy3-dye NHS Ester 1 tube Cy5 or Cy3 monofunctional reactive dye (Amersham, order # PA23001) 40 µl DMSO Store in 8 x 5 µl aliquots at 80°C up to 3 months Use 5 µl per labelling Toxic chemicals HYDROXYLAMINE (See Chemical safety cards on http://kluyver/intranet/) Chemical dangers: May explode on heating above 70°C or when exposed to open flame. The substance decomposes rapidly at room temperature, especially in presence of moisture and carbon dioxide, and violently on heating producing toxic fumes including nitrogen oxides. The solution in water is a weak base. Reacts violently with oxidants, metals such as finely divided zinc, some metal oxides, copper(II)sulfate and phosphorus chlorides causing fire and explosion hazard. Effects of short-term exposure: The substance irritates the skin and the respiratory tract, and is corrosive to the eyes. The substance may cause effects on the blood, resulting in formation of methaemoglobin. The effects may be delayed. Medical observation is indicated. Effects of long-term or repeated exposure: Repeated or prolonged contact may cause skin sensitization. The substance may have effects on the blood, resulting in formation of methaemoglobin and the consequent anaemia.
Hybridization protocol
Application This protocol can be used for hybridisation of Cy3/Cy5 labelled cDNA to 11K Agilent slides. It is an abbreviated form of, and an addition to the manufacturer's protocol (also available as pdf in BASE under "protocols" as "Agilent hyb. protocol v 4.1"). All chemicals mentioned in the hybridisation kit are supplied in the Agilent in situ hybridisation kit-plus (Order# 5184-3568, lab Arno, room temperature). Micro array slides, hybridisation chambers and gasket slides (Order# G2534-60002) are available in Arno's lab. 20x SSC (see NIZO QA T/BFC063) is needed in the wash solutions and has to be made prior to the washing steps. Hybridisation Tips When the volume of the required amount of labelled cDNA exceeds 40 µl evaporate the excess of liquid in the SpeedVac (reduction from 120 to 40 µl takes about 45 min without heating) Be very sure that you make 210 µl of hybridization mix, otherwise the array chamber will not be filled to at least 50%. This will cause a "black hole" in the centre of the array image. In case you conclude that you do not have enough hybridization mix you can add some hybridization buffer (mix of equal volumes water and 2x hybridization buffer). Once the chamber is closed, mark the level of buffer on the gasket slide (the one with the rubber rings) using a fine-liner. This allows you to check afterwards whether leakage has taken place. Cy-dye bleaching has been shown to be promoted by light, ozone and humidity (relevant for dried slides). On hot summer days, humid days or in the vicinity of UV lamps dyes may bleach very quickly. Hybridisation Special powder free gloves are available for working with slides. Avoid too much light during the procedure to prevent the cyanine dyes from bleaching. 1. Take the necessary number of Agilent slides from the desiccator and close the desiccator properly. Notate the serial numbers in your journal. The side containing the bar code and "Agilent" label contains the spots, the side with the bar code and the slide number label is the back. The array that is furthest away from the label is array number 1, the closest is array number 2 (see Figure 1). 2. Prepare 0.3 g of Cy3 and 0.3 g of Cy5 labelled cDNA's as described in "cDNA synthesis and labelling" each in a volume of 40 µl (see tips if concentration is too low, or add water if concentration is too high. Also see note below) 3. Mix the Cy3 and Cy5 labelled cDNA's in an amber Eppendorf tube 4. Heat at 98 °C for 3 minutes and cool down to room temperature (use clamp or pierce a hole in the lid to avoid spontaneous opening of the lids when heating) 5. Add 25 µl of 10x control (freezer, Arno) 6. Add 105 µl 2x hybridisation buffer (hybridisation kit) The total volume will now be 210 µl 7. Mix (no vortex to avoid bubble formation) and spin briefly to collect all of the mix at the bottom of the tube 8. Put a gasket slide (G2534-60002) in the microarray hybridisation chamber (G2534A) and pipet the hybridisation mixtures in the centre of the array frames without introduction of bubbles 9. Put the microarray slide slightly on top of it without introduction of bubbles (the side containing the bar code and "Agilent" label has to face towards the hybridation mixture). The easiest way to achieve this is to put one short side of the microarray slide on the gasket slide and slowly lower the array slide 10. Place the upper part of the hybridisation chamber (chamber cover) and the chamber clamp and close this by turning the thumbscrew clockwise and tighten it firmly by hand (see Figure 2) Do not use tools to tighten the thumbscrew when closing the hybridisation chamber!! 11. Take the hybridisation chamber and turn it vertically to check for correct bubble formation. Bubbles are correct when they move freely in the hybridisation chamber and the mixture can reach the complete surface of the slide upon turning of the slide. Also check whether the chamber is filled to at least 60% of its total volume 12. Hybridise for 17 hours at 60 °C. If necessary balance the rotor with empty hybridization chambers Note about the amount of cDNA to be used per array We are still discussing whether the amount of cDNA to be used on a microarray should be normalized with respect to the concentration of cDNA measured in the sample or with respect to the concentration of Cy-dye measured. Amersham recommends the second option. However, until now we have used the first option (0.3 g cDNA per array). Until now we have observed ratios of Cy-dye/cDNA of 0.03 to 0.12 pmol/ng yielding good final results. Washing Tips Before starting the washing steps prepare all washing solutions and put them in washing trays (lab Arno) If more than one slide has to be washed, separate all array and gasket slides as described and keep the array slides submerged in solution 1 until all slide sandwiches are separated. Continue with the washing steps for all slides at the same time. To avoid exposure to light place a box over the washing trays when washing the slides. Preparation of wash solutions Wash solution 1 (6x SSC, 0.005% Triton X-102) 700 ml milliQ 300 ml 20x SSC (NIZO-SOP T/BFC063) 0.500 ml 10% Triton X-102 (hybridization kit) Mix the components and pass the solution through a 0.22 µm filter Store at room temperature Wash solution 2 (0.1x SSC, 0.005% Triton X-102) 995 ml milliQ 5.0 ml 20x SSC (NIZO-SOP T/BFC063) 0.500 ml 10% Triton X-102 (hybridization kit) Mix the components and pass the solution through a 0.22 µm filter Store at 4 ºC Washing 1. After hybridisation remove the glass slide from the hybridisation chamber and separate the glass slides completely submerged in wash tray 1 containing wash solution 1 at room temperature by inserting the blunt tip of the plastic tweezers at the bar code end of the slide sandwich and gently turning the tip of the tweezers. 2. Transfer the slides to wash tray 2 containing wash solution 1 and a magnetic stirrer and place this on a magnetic stir plate. Wash the slides for 10 minutes at room temperature at medium stirring. 3. Transfer the slides to wash tray 3 containing pre-cooled wash solution 2 and a magnetic stirrer and place it in another tray filled with melting ice on a magnetic stir plate. Wash for 5 minutes at medium stirring. 4. Dry the slides by blowing all droplets of washing solution to the edges of the slides using a nitrogen airgun (Arno). Adjust the manometer to 1 atm. 5. Put the slides in a dark dust-free box (microscope slide box, at the reader) and scan the slide immediately (see protocol "Scanning of DNA microarrays"). Figure 1. Our convention for numbering the arrays on an Agilent slide. Figure 2. Turning the thumbscrew hand-tight.
Scan protocol
Application This procedure can be followed to scan DNA micro array slides on the Scan Array Express microarray scanner. Operating the array scanner 1. Turn on the scanner and the computer (in this order) 2. Login: scanner, password: scanner2 3. double click the 'ScanArray Express' icon 4. Switch on lasers 1 and 3 at least 15 minutes prior to scanning arrays 5. Put focus on file This is to prevent you from accidentally turning of the laser again after the 15 minutes warming-up phase 6. Press 'Scan | Prescan' 7. Put resolution at 50 nm, and PMT value for both channels at for instance 70% (spotted arrays) or 40% (Agilent arrays) 8. Press 'Start' 9. Press 'Palette,' 'Green' as soon as projection of the image has started (first dye), select the red colour as soon as scanning of the second layer (second dye) has started 10. Select frame for high resolution scan, adjust resolution (to 10 nm) and scan the array for both dyes. Adjust PMT values to balance signals obtained for both channels Balance both channels as well as possible to achieve a comparable average intensity for both channels. This avoids large normalisation corrections during analysis of the data, so decreases the chance on introduction of artefacts. Make at least one scan in which none of the spots are saturated. Run this scan at for instance PMT = 40% and step-wise increase the PMT level until both channels have comparable intensities (e.g. 1000 intensity units) 11. Press 'File | Save' to save the files, 'Save all' saves both dye layers Create a new folder for each array 'Save all' saves the signals from both layers in individual files It's possible to save the 'composite' file in .jpg format, however this is not necessary since al data are in the original files as well 12. Switch off the lasers and the scanner after use Tips Allow the lasers to warm for at least 15 minutes Quantification of spots 1. Start Imagene by double clicking the 'Imagene' icon 2. Load tiff files of both channels 3. Load the template file from the folder 'GAL files' located on the desktop Every batch of arrays has it's own GAL file. The name of the GAL file corresponding to the batch of slides can be found under 'Array LIMS | Array designs' as 'print map/reporter map' in BASE (http://kluyver/base). (e.g. LpGn07.gal for the several batches of spotted L. plantarum arrays from Groningen and '012731_D_20040830.gal' that was edited to lpag01.gal for the 1st design of Agilent L. plantarum slides) 4. Adjust frame by pressing the 'Rectangle adjust' button, drag the circles on the spots, starting with the left-up spot (this is not necessary for Agilent slides since they are recognised automatically) 5. Press the 'Automatically place grid' button and check by eye if the grid was placed correctly 6. Press 'Quantify' (a table with spot id's and intensities is produced) 7. Save files (for both dyes) in the same directory as the original tiff files are located just by selecting (single click) the directory (don't open!) and press the 'Save' button 8. Remove slides by pressing 'File,' 'Remove selected images' for both channels before loading and quantification of other arrays (only necessary when more slides are to be quantified) Repeat steps 14, 16-20 to load and quantify multiple slides after the current slides have been removed. Repeat step 15 when another type/batch of slides is used. 9. Import data in BASE (http://kluyver/base) Although the choice is completely arbitrary, for clarity always use channel 1 for the Cy3 label and channel 2 for the Cy5 label to avoid accidental swapping of labels during data analysis.
Description
In these experiments we compare the gene expression of the Lactobacillus plantarum WCFS1 harbouring the control plasmid pNZ7021. This plasmid is a high copy vector and contains a chloramphenicol marker, a multiple cloning site (to insert genes), prior to the multiple cloning site the pepN promoter is located, this promoter is a strong promoter of the lactic acid bacterium Lactoccus lactis. The strain to which the gene expression is compared is Lactobacillus plantarum WCFS1 harboring plasmid pNZ7026, this plasmid contains the complete folate gene cluster of L. plantarum WCFS1. The folate genes on these plasmid have the following names while present on the chromosome of L. plantarum WCFS1, Lp_3294, Lp_3294, Lp_3295, Lp_3296, Lp_3297, Lp_3298 and Lp_3299. In short, we compare the gene expression of the control strain of L. plantarum with a L. plantarum strain that can produce high amounts of folate. Both strains were cultivated in quadruple on chemically defined medium as described by Teusink et al AEM 2005, we have used in this experiment a glucose concentration of 25mM and the total amount of para-aminobenzoicacid that used is 10mg/l. The type of cultivation we used was chemostat, after the strains were grown in batch the medium-in and medium-out pumps were turned on and thereby a growth rate of 0.35 h-1 was used. In steady state we have harvested RNA and the RNA was handled as described in this protocol. As stated above, the control and folate overproducing L. plantarum strains were cultivated 4 independent times in chemostat experiments. The names that were given to the RNA from the control experiment were W2, W3L, W3R and Warno; alternatively, the names that were give to the RNA from the folate overproducing strain were called Hy2 (named after hyper), Hy1L, Hy1R and Hyparno. We have used the following hybridisation scheme to compare the samples with each other. W2 and Hy2, Hy2 and W3L, W3L and Hy1L, Hy1L and W3R, W3R and Hy1R, Hy1R and W2, W2 and W3R, Hy1L and Hy2, Warno and Hyparno, W3R and Warno, Hyparno and Hy2, Hyparno and W3L.
Data processing
After blank spots had been removed, array measurements were normalized by local fitting of an M-A plot applying the loess algorithm, using the Limma package in R (http://www.r-project.org). Spot intensities in both channels (I1 and I2) were thus converted to M-A coordinates, where M = log2(I1/I2) and A = log2(I1/I2)/2. Normalized intensities were used for further analysis. The statistical significance of differences was calculated from variation in biological replicated, using the eBayes function in Limma (cross-probe variance estimation) and Holmes determination of significance. Only genes with a log2 ratio of -1 and +1 and a Holmes value less than 0.1 were considered significant.
