Human trabecular meshwork (TM) cell lines treated with 0.0025% benzyl alcohol (BA) for 12 hours.
Biomaterial provider
Dr. Thai Nguyen
Extracted molecule
total RNA
Extraction protocol
1. Disrupt cells by Buffer RLT: Add the appropriate volume of Buffer RLT. Vortex or pipet to mix. 2. Homogenize the sample: Pipet the lysate directly on to a QIAshredder spin column, centrifuge for 2 min at maximum speed (13,200 rpm). 3. Add 1 volume (350 ul or 600 ul) of 70% ethanol to the homogenized lysate, and mix well by pipetting. Do not centrifuge. 4. Apply up to 700 ul of the sample, including any precipitate that may have formed, to an RNeasy mini column placed in a 2-ml collection tube. Close the tube gently, and centrifuge for 15 sec at 10,000 rpm. Discard the flow-through (not compatible with bleach). If any, add remaining sample, repeat this step. 5. Add 700 ul Buffer RW1 to the RNeasy column. Close the tube gently, and centrifuge for 15s at 10,000 rpm. Discard the flow-through and collection tube. 6. Transfer the RNeasy column into a new 2-ml collection tube (supplied). Pipet 500 ul Buffer RPE onto the RNeasy column. Close the tube gently, and centrifuge for 15 sec at 10,000 rpm. Discard the flow-through. 7. Add another 500 ul Buffer RPE to the RNeasy column. Close the tube gently, and centrifuge for 2 min at 10,000 rpm to dry the RNeasy silica-gel membrane. 8. To elute, transfer the RNeasy column to a new 1.5-ml collection tube (supplied). Pipet 30 - 50 ul RNase-free water directly onto the RNeasy silica-gel membrane. Close the tube gently, centrifuge for 1 min at 10,000 rpm to elute. 9. If the expected RNA yield is > 30 ug, repeat the step 9 with a second volume of RNase-free water. Elute into the same collection tube.
Label
Cy3
Label protocol
1. Labelling first-strand cDNA with amino allyl-dUTP (in 200 ul tube): 1) Set a thermocycler at 70 C and another at 42 C. 2) Add the following components on ice (Primer annealing): RNA (25 ug) Anchored oligo (dT) (3 ul) Total volume (11 ul) 3) Mix gently by pipetting up and down. 4) Incubate reactions at 70 C for 5 min. 5) Cool reactions at room temperature for 10 min to allow the primers and the mRNA template to anneal. 6) Spin down reactions for 15 sec in a microcentrifuge. 7) Place reactions on ice and add the following components to each, adding the enzyme last. (Extension reaction) 5x CyScript buffer (4 ul) 0.1 M DTT (2 ul) Nucleotide mix (1 ul) AA-dUTP (1 ul) Cyscript reverse transcriptase (1 ul) Total volume (20 ul) 8) Mix by very gently pipetting and spin for 15 sec (Vigorous pipetting will denature the enzyme). 9) Incubate the reactions at 42 C for 90 min. 10) Store the aminoallyl modified cDNA on ice for immediate purification or place at -20 C frost freezer for storage. (Can stop) 2. Purification of cDNA with Cyscribe GFX Purification Kit: 1) Adjust a waterbath to 37 C. 2) Add 2 ul 2.5 M NaOH to each cDNA reaction. (Degradation of mRNA) 3) Mix and spin for 15 sec. 4) Incubate reactions at 37 C for 15 min. 5) Add 10 ul 2 M HEPES free acid to each reaction (Neutralize the reaction). 6) Mix by vortexing and spin for 15 sec. 7) The cDNA reactions are now ready for purification or can be stored at -20 C. (can stop) 8) Add 500 ul capture buffer to each GFX column. (Purification of amilnoallyl-labelled cDNA with CyScribe GFX Purification Kit) 9) Transfer the unpurified aminoallyl-labelled cDNA products (32 ul) into each GFX column, mix the cDNA by gently pipetting up and down 5 times. 10) Centrifuge each column immediately at 13000 rpm for 30 sec. 11) Remove the GFX column and discard the liquid at the bottom of each collection tube. Return each GFX column into the used collection tube. 12) Add 600 ul of 80% ethanol to each column and centrifuge at 13,000 rpm for 30 sec. 13) Remove the GFX column and discard the collected liquid. Repeat the 80% ethanol wash for a total of 3 washes. Discard the liquid and place each column back in the used collection tube. 14) Centrifuge at 13,000 rpm for an additional 10 sec to remove all traces of 80% ethanol in the tip of the column. Discard the collection tube. 15) Transfer each GFX column to a fresh 1.5 ml microcentrifuge tube and add 60 ul of 0.1 M NaHCO3 directly to the top of the glass fiber matrix in each GFX column (Make sure completely covering the membrane). 16) Incubate the GFX column at room temperature for 1-5 min. Centrifuge at 13,000 rpm for 1 min to collect the purified aminoallyl-labelled cDNA. 17) Repeat steps 15 and 16 may increase the yield by 10%. 18) Proceed immediately to the coupling reaction procedure. 3. Post-labelling of amino allyl-modified cDNA with CyDye: 1) Add the purified aminoallyl cDNA directly into one aliquot of CyDye NHS ester in a 1.5 ml tube. Resuspend the NHS ester completely by pipetting several times. Centrifuge at 13,000 rpm for 1 min to collect the liquid at the bottom of the tube. 2) Incubate at room temperature in the dark for 60-90 min. 3) Add 15 ul of 4 M hydroxylamine to each coupling reaction to stop the reaction. 4) Pipette up and down several times to mix the components, and then incubate at room temperature in the dark for 15 min. 5) Purify the CyDye-labelled cDNA immediately according to the following protocol. 4. Purification of CyDye-labelled cDNA: 1) Add 40 ml of absolute ethanol to the wash buffer bottle prior to starting the protocol. Mix well. 2) Add 500 ul of capture buffer to each GFX column. 3) Transfer the unpurified labeled cDNA products into each GFX column, mix the cDNA by gently pipetting up and down 5 times. 4) Centrifue each column immediately at 13,000 rpm for 30 sec. 5) Remove the GFX column and discard the liquid. Return each GFX column to the used collection tube. 6) Add 600 ul of wash buffer to each column and centrifuge at 13,000 rpm for 30 sec. 7) Remove the GFX column and discard the collected liquid. Repeat the wash buffer wash for a total of 3 washes. After the final wash, discard the liquid and place each column back in the used collection tube. 8) Centrifuge each column at 13,000 rpm for an additional 10 sec to remove all wash buffer in the tip of the column. Discard the collection tube. 9) Transfer each GFX column to a fresh 1.5ml microcentrifuge tube , add 60 ul of prewarmed (65 C) elution buffer directly to the top of the glass fiber matrix in each GFX column (Make sure completely covering the membrane). 10) Incubate the GFX column at room temperature for 1-5 min. Centrifuge at 13,000 rpm for 1 min to collect the purified labeled cDNA. 11) Repeat steps 9 and 10 may increase the yield by 10%. 12) Measure the purified cDNA in a spectrophotometer at 260nm (Cy5 at 649nm and Cy3 dye at 550nm) to calculate the yield of purified cDNA. Incorporation of CyDye = pmol of CyDye in sample/ ug of nucleic acid in sample Typical yield of labeled cDNA with the recommended purification systems varies from 40 – 100 pmol of Cy3 or Cy5 incorporated into probe using 0.5 ug control mRNA as template. Frequency of Incorporation (FOI) = pmol of Dye incorporated x (324.5/ng of cDNA probe). Probes with FOI values in the range of 20-50 generate strong signals upon hybridization. FOI value <10 will generate weak signals from poor incorporation, whereas, at FOI >70, probes may also produce reduced hybridization signals from possible quenching between CyDyes fluors.
Human trabecular meshwork (TM) cell lines treated with 0.1 mg/ml triamcinolone acetonide (TA) for 12 hours.
Biomaterial provider
Dr. Thai Nguyen
Extracted molecule
total RNA
Extraction protocol
1. Disrupt cells by Buffer RLT: Add the appropriate volume of Buffer RLT. Vortex or pipet to mix. 2. Homogenize the sample: Pipet the lysate directly on to a QIAshredder spin column, centrifuge for 2 min at maximum speed (13,200 rpm). 3. Add 1 volume (350 ul or 600 ul) of 70% ethanol to the homogenized lysate, and mix well by pipetting. Do not centrifuge. 4. Apply up to 700 ul of the sample, including any precipitate that may have formed, to an RNeasy mini column placed in a 2-ml collection tube. Close the tube gently, and centrifuge for 15 sec at 10,000 rpm. Discard the flow-through (not compatible with bleach). If any, add remaining sample, repeat this step. 5. Add 700 ul Buffer RW1 to the RNeasy column. Close the tube gently, and centrifuge for 15s at 10,000 rpm. Discard the flow-through and collection tube. 6. Transfer the RNeasy column into a new 2-ml collection tube (supplied). Pipet 500 ul Buffer RPE onto the RNeasy column. Close the tube gently, and centrifuge for 15 sec at 10,000 rpm. Discard the flow-through. 7. Add another 500 ul Buffer RPE to the RNeasy column. Close the tube gently, and centrifuge for 2 min at 10,000 rpm to dry the RNeasy silica-gel membrane. 8. To elute, transfer the RNeasy column to a new 1.5-ml collection tube (supplied). Pipet 30 - 50 ul RNase-free water directly onto the RNeasy silica-gel membrane. Close the tube gently, centrifuge for 1 min at 10,000 rpm to elute. 9. If the expected RNA yield is > 30 ug, repeat the step 9 with a second volume of RNase-free water. Elute into the same collection tube.
Label
Cy5
Label protocol
1. Labelling first-strand cDNA with amino allyl-dUTP (in 200 ul tube): 1) Set a thermocycler at 70 C and another at 42 C. 2) Add the following components on ice (Primer annealing): RNA (25 ug) Anchored oligo (dT) (3 ul) Total volume (11 ul) 3) Mix gently by pipetting up and down. 4) Incubate reactions at 70 C for 5 min. 5) Cool reactions at room temperature for 10 min to allow the primers and the mRNA template to anneal. 6) Spin down reactions for 15 sec in a microcentrifuge. 7) Place reactions on ice and add the following components to each, adding the enzyme last. (Extension reaction) 5x CyScript buffer (4 ul) 0.1 M DTT (2 ul) Nucleotide mix (1 ul) AA-dUTP (1 ul) Cyscript reverse transcriptase (1 ul) Total volume (20 ul) 8) Mix by very gently pipetting and spin for 15 sec (Vigorous pipetting will denature the enzyme). 9) Incubate the reactions at 42 C for 90 min. 10) Store the aminoallyl modified cDNA on ice for immediate purification or place at -20 C frost freezer for storage. (Can stop) 2. Purification of cDNA with Cyscribe GFX Purification Kit: 1) Adjust a waterbath to 37 C. 2) Add 2 ul 2.5 M NaOH to each cDNA reaction. (Degradation of mRNA) 3) Mix and spin for 15 sec. 4) Incubate reactions at 37 C for 15 min. 5) Add 10 ul 2 M HEPES free acid to each reaction (Neutralize the reaction). 6) Mix by vortexing and spin for 15 sec. 7) The cDNA reactions are now ready for purification or can be stored at -20 C. (can stop) 8) Add 500 ul capture buffer to each GFX column. (Purification of amilnoallyl-labelled cDNA with CyScribe GFX Purification Kit) 9) Transfer the unpurified aminoallyl-labelled cDNA products (32 ul) into each GFX column, mix the cDNA by gently pipetting up and down 5 times. 10) Centrifuge each column immediately at 13000 rpm for 30 sec. 11) Remove the GFX column and discard the liquid at the bottom of each collection tube. Return each GFX column into the used collection tube. 12) Add 600 ul of 80% ethanol to each column and centrifuge at 13,000 rpm for 30 sec. 13) Remove the GFX column and discard the collected liquid. Repeat the 80% ethanol wash for a total of 3 washes. Discard the liquid and place each column back in the used collection tube. 14) Centrifuge at 13,000 rpm for an additional 10 sec to remove all traces of 80% ethanol in the tip of the column. Discard the collection tube. 15) Transfer each GFX column to a fresh 1.5 ml microcentrifuge tube and add 60 ul of 0.1 M NaHCO3 directly to the top of the glass fiber matrix in each GFX column (Make sure completely covering the membrane). 16) Incubate the GFX column at room temperature for 1-5 min. Centrifuge at 13,000 rpm for 1 min to collect the purified aminoallyl-labelled cDNA. 17) Repeat steps 15 and 16 may increase the yield by 10%. 18) Proceed immediately to the coupling reaction procedure. 3. Post-labelling of amino allyl-modified cDNA with CyDye: 1) Add the purified aminoallyl cDNA directly into one aliquot of CyDye NHS ester in a 1.5 ml tube. Resuspend the NHS ester completely by pipetting several times. Centrifuge at 13,000 rpm for 1 min to collect the liquid at the bottom of the tube. 2) Incubate at room temperature in the dark for 60-90 min. 3) Add 15 ul of 4 M hydroxylamine to each coupling reaction to stop the reaction. 4) Pipette up and down several times to mix the components, and then incubate at room temperature in the dark for 15 min. 5) Purify the CyDye-labelled cDNA immediately according to the following protocol. 4. Purification of CyDye-labelled cDNA: 1) Add 40 ml of absolute ethanol to the wash buffer bottle prior to starting the protocol. Mix well. 2) Add 500 ul of capture buffer to each GFX column. 3) Transfer the unpurified labeled cDNA products into each GFX column, mix the cDNA by gently pipetting up and down 5 times. 4) Centrifue each column immediately at 13,000 rpm for 30 sec. 5) Remove the GFX column and discard the liquid. Return each GFX column to the used collection tube. 6) Add 600 ul of wash buffer to each column and centrifuge at 13,000 rpm for 30 sec. 7) Remove the GFX column and discard the collected liquid. Repeat the wash buffer wash for a total of 3 washes. After the final wash, discard the liquid and place each column back in the used collection tube. 8) Centrifuge each column at 13,000 rpm for an additional 10 sec to remove all wash buffer in the tip of the column. Discard the collection tube. 9) Transfer each GFX column to a fresh 1.5ml microcentrifuge tube , add 60 ul of prewarmed (65 C) elution buffer directly to the top of the glass fiber matrix in each GFX column (Make sure completely covering the membrane). 10) Incubate the GFX column at room temperature for 1-5 min. Centrifuge at 13,000 rpm for 1 min to collect the purified labeled cDNA. 11) Repeat steps 9 and 10 may increase the yield by 10%. 12) Measure the purified cDNA in a spectrophotometer at 260nm (Cy5 at 649nm and Cy3 dye at 550nm) to calculate the yield of purified cDNA. Incorporation of CyDye = pmol of CyDye in sample/ ug of nucleic acid in sample Typical yield of labeled cDNA with the recommended purification systems varies from 40 – 100 pmol of Cy3 or Cy5 incorporated into probe using 0.5 ug control mRNA as template. Frequency of Incorporation (FOI) = pmol of Dye incorporated x (324.5/ng of cDNA probe). Probes with FOI values in the range of 20-50 generate strong signals upon hybridization. FOI value <10 will generate weak signals from poor incorporation, whereas, at FOI >70, probes may also produce reduced hybridization signals from possible quenching between CyDyes fluors.
Hybridization protocol
1. For dual color hybridization, combine Cy3- and Cy5-labelled cDNAs into one tube. Dry down the cDNA solution using MicroconYM-30. 1) Insert Microcon sample reservoir into vial. 2) Spin-rinse devices with 25 ul deionized water at 3000 rpm for 2 min. 3) Pipeette 120 ul combined labeled cDNA into sample reservoir, without touching the membrane with the pipette tip. Seal with attached cap. 4) Place assembled device into the centrifuge rotor, align the cap strap toward the center of the rotor. Spin at 8,000 rpm for 8 min. 5) Remove assembly from centrifuge. Separate vial from sample reservoir. 6) Place sample reservoir upside down in a new vial, spin at 3,300 rpm for 3 min. 2. Measure the obtained cDNA volume ( ~ 15 ul). 3. Denature the cDNA by heating at 95 C for 2 min. Leave the tube cap open to further dry the solution to 10.5 ul. 4. Cool the cDNA solution on ice for 30 sec. 5. Add 1.75 ul dA80 (1 mg/ml) and mix well. 6. Incubate the mixure at 75 C for 45 min. 7. Add 8.75 ul microarray hybridization buffer and 14 ul 100% Formamide. Mix well and spin for 15 sec in a microcentrifuge to collect all reaction components at the bottom of the tube. cDNA solution 10.5 ul (30%) dA80 (1 mg/ml) 1.75 ul (5%) 4x Hybridization buffer 8.75 ul (25%) 100% Formamide 14 ul (40%) Total volume 35 ul (100%) 8. Pipette the hybridization mixture onto a microarray slide. Carefully place the coverslip on the array. Avoid the formation of air bubbles under the coverslip. 9. Hybridize at 42 C for 16 hours in a humid hybridization chamber. Protect the slides form light during this and subsequent steps.
Scan protocol
Scan the slides with a microarray scanner suitable for detecting Cy3 and Cy5 fluorescence. 1. Lunch ScanArray software, open Lamp1 and Lamp 3, allow warming for 15 min. 2. Quick scan to find out suitable settings for laser power and gain (usually both 80%). 3. Scan with the highest resolution at 5 um. 4. Save both Cy3 and Cy5 images in a TIFF format. 5. Lunch QuantArray software, open Cy3 and Cy5 images to check the quality and process images.
Description
The first slide for TA-0.1 experiments.
Data processing
Print-tip-loess normalization method and quantile normalization method
