Protocol name: Microarray protocol with aminoallyl label
Prepared by: Monica Ponder
Date Created/Revised: 12/01/03
Purpose: Extraction of total RNA, cDNA
synthesis and Cy dye labeling for gene
expression studies
Reference(s): adapted from TIGR standard
protocol for aminoallyl labeling of RNA by Jeremy
Hasseman revision 2
Comments:
Using Strategene SpotReport Oligo array validation system
48°C is optimized temperature
Cell Preservation
1. Cells are grown under the desired condition to an OD that is midway in exponential
under both conditions using 325 mls of media in four 500ml Erylenmeyer flasks.
2. 25mls of culture were removed and while the swirling culture flask and combined. The
remaining 1150mL of culture was spun down for proteomics.
3. Cells are harvested by decanting into a sterile centrifuge bottle and adding a third volume
of RNAlater (chill for low temperature experiments).
4. Centirfuge under same temperature conditions in Sorvall at 10,000 x g for 10 minutes.
5. Make mixture of 10mls of DEPC treated 1xPBS and 3mls of RNAlater.
6. Decant supernatant and resuspend pellet in 3:1 mix of sterile 1xPBS/RNAlater. If using
40 ml volume resuspend in 3mls, will give you 13.3ml volume of total culture and 7.5mls
if using larger centrifuge bottle.
7. Place 1ml of RNAlater/cell mix in sterile 1.5 ml nuclease free eppi tubes and centrifuge
to collect cells. Place at -20°C until ready to use.
mRNA extraction protocol:
1. Decant RNAlater used to preserve pelleted cells.
2. Wash cells in 1ml RNase free water and spin at 10,000rpm for 5 minutes to collect cells.
Decant supernatant.
3. Use 100ul of 400ug/ml lysozyme in TE buffer, incubate at 37°C for 30 minutes to
degrade cell wall.
4. Follow protocol for bacteria from the Qiagen RNeasy kit.
Amplification protocol:
1. Prepare mRNA from spot array kit. Make mix of 1uL of each.
Oligo#
Amount for 2ug total RNA
Dilution for 1ul
1
1ng
2 ul stock to 20uL H20
2
500 pg
2 to 40uL
3
250pg
2 to 80uL
4
125pg
2 to 160 ul
5
62.5pg
2 to 320ul
6
31.25 pg
2 to 640ul
7
15.6 pg
2 to 1280ul
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2.
3.
4.
5.
8
7.81 pg
1 to 1280ul
9
3.4 pg
0.5 to 1280
10
1.2 pg
0.25 to 1280
2 ug total RNA, 2 ul random primers and enough water for final volume of 18.5ul
combined in nuclease free tube (we use less RNA because get same cDNA yield as
with 10ug)
Place at 70°C for 10 minutes to allow for the random primers to complex with the
RNA.
Shift tube immediately to ice/-80°C ETOH bath so the RNA will remain bound to
the random primers for 1 minute. Should see pellet freeze. Allow pellet to thaw
before adding
The reverse transcription mixture is made from (per reaction):
6ul
5x Superscript buffer
3ul
0.1M DTT
2ul
Superscript II RT enzyme
1.2 ul
25mM aa-dNTP mix (5ul 100mM dATP,dCTP,dGTP
2ul 100mM dTTP, 3ul aa-dUTP)
(we use 2:3 dTTP/aa-dUTP ratio due to low GC content)
Also don’t thaw the aaUTP mix more than twice or yield will be low!
Prepare a master mix and add 12.2 ul denatured RNA/ primer mix. Mix by pipetting.
incubate at 42°C overnight .
6.
7.
Hydrolyze the RNA by adding 10ul of 1N NaOH and 10ul of 0.5M EDTA. Place at
65°C for 15 minutes.
Follow by addition of 25ul 1M Tris-HCl pH 7. Mix by pipetting. The free amines
from the Tris should not interfere with labeling because it will be removed in the
purification step.
cDNA clean-up
*use modified Qiagen PCR purification
1. Added 5 volumes (317.5uL) of buffer PB to cDNA immediately after adding Tris.
Mix by pipetting. Load entire volume onto spin column and spin at room temperature
14,000 x g for 1 minute. Decant eluent.
2. Mix phosphate wash buffer by vortexing. Wash columns twice with 750 ul of
phosphate wash buffer (0.5mL 1M KPO4 , 15.25mL MilliQ, 82.25mL 95%ETOH)
under same spin conditions.
3. Spin one more time 1 minute to completely remove the residual ethanol.
4.
Transfer column to new amber eppi tube and add 30 ul of phosphate elution buffer
(4mM KPO4). Allow to sit at room temp for 1 minute before placing in centrifuge to
spin at 14,000 x g for 1 minute. Repeat for total elution volume of 60 ul.
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5.
A cDNA gel can be ran to assure quality of cDNA by denaturing the single stranded
cDNA at 70°C for 10 minutes and then loading into a 1% agarose gel. If the cDNA is
not degrading then should see a streak from about 500 KB. If degraded all products
will be smaller.
6. Dry the cDNA in the speed vac under medium heat about 20-30 minutes.
Incorporating the SPOTarray kit
Labeling: amino allyl
1. Resuspend the cDNA in 4.5ul of fresh-2 week old 0.1M sodium carbonate (5.4g
Na2CO3 per 40mL MilliQ bring to pH 9 with HCl and bring up to volume with
MillIQ. Dilute stock 1:10 for final concentration). Sodium carbonate can also be
kept at -20°c but must check the pH. Add 5uL HCl to 1mL to return pH10 to pH9.
2.
Add 4.5 uL of dye. Dye should be stored in air tight jar at -80°C so that the DMSO
doesn’t absorb water. Dye should be resuspended in 73ul of DMSO.
3.
Incubate at 25°C in the tropi-cooler (needs to be stable) with foil on the top 1-2
hours.
4.
Added 35ul 0.1M NaOAc pH 5.2
5.
Purify the coupled dye-CDNA with Qiagen PCR purification kit by standard
procedure. Elute twice with 30 ul of elution buffer. Should see a lot of the dye on
the filter when there is a good incorporation rate.
6.
7.
Measure the OD 260, 550 and 650 of the entire sample (60uL).
Calculate the:
amount of CDNA synthesized (pmol)= (OD 260*37*volume*1000)/324.5
pmol Cy3= OD 550* volume/ 0.15
pmol Cy5= OD 650*volume/ 0.25
dye incorporated per nt= pmol cDNA/ pmol dye
**For hybridization should have less than 50 nucleotides/dye and more than 200 pmol
dye for good signal
8.
Mix equal amounts of Cy5 and Cy 3 labeled cDNA that will be hybridized to same
slide and dry in speed vac. Adjust so all slides will have equal pmol dye and
hopefully close in cDNA. Make sure covered in foil.
Hybridization
CMT ULTA GAPS slides (Corning):
Pre-hybridization step:
1. UV crosslinking of the slides at 120 mJ performed by Jeff after printing.
2. Prepare pre- hybridization solution of 5X SSC, 0.1% SDS, 0.1% BSA and filter
sterilize. Preheat at 42°C for 30 minutes before use.
3. Place slides in coplin Jar with pre- hyb solution at 50°C for 45 minutes.
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4. Wash slides by dipping in MilliQ 4-5 times.
5. Transfer to fresh water and dip 4-5 times this time only submerging printed area.
6. Next dip the slides in isopropanol.
** Important don’t let the slide dry out or will streak**
7. Dry slides in the centrifuge at lowest speed for 5 minutes. Look for streaking or film
appearance. If this is seen repeat the water and isopropanol washes. Dry Lifterslips
under nitrogen stream.
8. Place slides in the Telechem hybridization chambers with 50 uL of 3X SSC (10uL if
using corning) added to maintain humidity. Lifter slip placed on the slide.
9. Place slide in the hybridization oven at 48°c to preheat. Reduces bubbles and
background.
**Pre-hyb should be finished no sooner than 15 minutes before hybridization takes place
or background levels increase**
Hybridization
1. Preheat hybridization buffer to desired temperature (48°C).
2. Resuspend the labeled cDNA in 7ul of MilliQ
3. Add 2uL of herring sperm DNA (10 mg/ml)
4. Place at 96°C for 3 minutes to denature
5. Mix 52ul of Glass Hyb buffer (Clonetech) with the denatured cDNA.
6. Preheat to 48°
7. Apply to the slide at edge of coverslip nearest the printed portion of array and
allow capillary action to evenly distribute the hybridization solution.
*Use narrow tips or narrow gauge needle so that can get under coverslip easier*
8. If using Telechem chambers tighten the hyb chamber screws using
thumbsrews and screwdriver or less will see humidity gradients.
9. Place the slides in shaking water bath in direction of motion (horizontal) or
gradients of background will be visible on slide. Incubate at 48°C in gently
shaking water bath for 16 hours.
Post-hybridization
Each wash 5 minutes shaking in pre-heated wash buffers at 48.5°C
1. Quick dip into pre-heated low stringency wash: 1X SSC, 0.1%SDS to remove
coverslip
2. Low stringency wash: 1X SSC, 0.1%SDS
3. Medium stringency wash: 0.1x SSC, 0.1%SDS
4. Medium stringency wash: 0.1x SSC, 0.1%SDS
5. High stringency wash: 0.05x SSC
6. Dip once in MillQ to remove excess SSC and spin to dry in centrifuge.
** Important don’t let the slide dry out or will streak**
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