Affymetrix Labeling Protocols: Day1
First Strand Synthesis:
1. Add 5 ug total RNA
Primer
DEPC water
x ul
1 ul
y ul
10 ul
2. Incubate at 70C for 10 min.
Spin and put on ice.
3. Add 5X first strand buffer
0.1M DTT
2 ul
10 mM dNTP mix
1 ul
4 ul
4. Incubate at 42C for 2 min.
5. Add Superscript II (200 U/ul from kit) 1ul/5ug total RNA or 1 ug mRNA.
Total volume will be 20 ul
6. Incubate 1 hour at 42C.
7. Place on ice.
Second Strand Synthesis:
1. Add DEPC water
5X second strand buffer
10mM dACTGs
E. coli 10 U/ul DNA ligase
E. coli 10 U/ul DNA polymerase I
E. coli 2 U/ul RNase H
91 ul
30 ul
3 ul
1 ul
4 ul
1 ul
2. Mix gently, centrifuge and incubate at 16C
(Reagent Shelf)
2 hours.
3. Add 2ul (10 U) T4 DNA polymerase
4. Incubate at 16C for 5 min.
5. Add 10 ul 0.5 M EDTA (Reagent Shelf)
6. Store at –20C or continue to cleanup.
Cleanup of Double Stranded DNA:
1. Pellet Phase-Loc gel at 12,000x g for 30 sec.
2. Add 162 ul (equal volume) of Phenol Chloroform Isoamyl (25:24:1 pH
7.9/8.0) to final volume of 324 ul.
3. Vortex and transfer to PLG tubes.
4. Do Not Vortex again.
Centrifuge at 12,000x g for 2 min.
5. Transfer aqueous upper phase to 1.5 ml tube.
6. Add 0.5 vol. 7.5 M Ammonium Ac + 2.5 vol. 100% ethanol (-20) to sample
and vortex. (optional: add 1 ul (5mg/ml) glycogen as carrier).
Precipitate overnight at –20C.
Affymetrix Labeling Protocols
Day 2
cDNA Cleanup (continued)
7. Centrifuge at 12,000x g for 20 min. at room temp.
8. Remove supernatant. Wash with 80% ethanol (-20).
9. Centrifuge at 12,000x g for 5 min.
10. Remove ethanol and repeat steps 8 & 9 once more.
11. Air dry pellet and resuspend in 12 ul
DEPC water.
12. Store at –20C or continue.
RNA Labeling (IVT)
1. Determine volume of sample to give ~1 ug cDNA for template** check
with Affy:
Original total RNA (ug)
resuspension vol
5.0-8.0
8.1-16.0
16.1-24.0
24.1-32.0
32.1-40.0
Volume cDNA for IVT
**based on 12 ul
10 ul
5 ul
3.3 ul
2.5 ul
2 ul
2. Add in order to new microfuge tube:
Template cDNA
x ul
DEPC water
y ul (22 – x)
10X HY Reaction Buffer (Vial 1)
4 ul
10X Biotin labeled Ribonucleases (Vial 2)
4 ul
10X DTT (Vial 3)
4 ul
10X RNase Inhibitor Mix (Vial 4)
4 ul
20X T7 RNA Polymerase (Vial 5)
2 ul
40 ul total volume
3. Mix reagents carefully and spin briefly.
4. Incubate at 37C for 4-5 hours, mixing every 30-45 min.
5. Clean-up immediately or store at -20C or -70C
Cleaning Up IVT Products
1. Purify only one-half of IVT product initially (20 ul), then check
yield. Save aliquot of unpurified product for gel electrophoresis before
purifying other half.
2. Add 80 ul RNase-free water to 20 ul of IVT product for final volume of
100 ul.
3. Add 350 ul Buffer RLT and mix thoroughly.
4. Add 250 ul ethanol (96-100%) and mix well by pipetting. Do not
centrifuge.
5. Apply sample to an RNeasy mini spin column in a collection tube.
Centrifuge for 15 sec at ?8000 g.
6. Return flow-through to spin column and centrifuge again for 15 sec at
?8000 g. Discard second flow-through (not compatible with bleach).
7. Transfer spin column to new 2 ml collection tube. Add 500 ul Buffer
RPE and centrifuge for 15 sec at ?8000 g. Discard flow-through and reuse
collection tube.
8. Add 500 ul Buffer RPE to spin column and centrifuge for 2 min at max
speed to dry membrane. Optional: Place spin column in new collection tube
and centrifuge at max speed for 1 min to eliminate any possible carryover
of Buffer RPE.
9. Transfer spin column to 1.5 ml collection tube and pipette 30-50 ul of
RNase-free water (warmed) onto spin column membrane. Wait 1 min and
centrifuge for 1 min at ?8000 g to elute cRNA. Repeat if expected cRNA
yield is >30 ug (expected yield could be 50 ug from 0.5 ug cDNA).
10. Quantify cRNA concentration and purity before purifying second half
of IVT product.
Quantifying the Purified cRNA
1. Check OD at 260 nm and 280 nm. Need A260/A280 ratio between 1.9-2.1
2.0 for acceptable purity.
2. Calculate cRNA yield:
cRNA (ug) = A260 * 40 ug/ml * [spec vol (ul) / IVT product in spec
(ul)] * elution vol (ml)
3. Calculate adjusted cRNA yield if started with total RNA:
Adjusted cRNA yield = RNAm – (total RNAi)(y)
RNAm = amount of cRNA measured after IVT (ug)
total RNAi = starting amount of total RNA (ug)
y = fraction of cDNA reaction used in IVT
4. Determine overall concentration of adjusted cRNA product from both
halves of purified IVT produced. If cRNA concentration is < 0.6 ug/ul,
precipitate sample and resuspend in appropriate volume of RNase-free
water.
Ethanol Precipitation
1. Add 0.5 volumes NH4Ac to purified cRNA sample.
2. Add 2.5 volumes (sample vol + NH4Ac) of absolute ethanol (-20C) and
vortex. Optional: add 1 ul glycogen to help precipitate and visualize
cRNA.
3. Precipitate at -20C overnight.
Affymetrix Labeling Protocols
Day 3
Ethanol Precipitation (cont.)
1. Spin precipitated sample at ?12,000 g at 4C for 30 min.
2. Wash pellet twice with 500 ul of 80% ethanol (-20C). Air dry pellet
before resuspension.
3. Resuspend dried pellet in appropriate volume of RNase-free water.
Final concentration (adjusted) of cRNA should be at least 0.6 ug/ul.
Fragmenting the cRNA
1. Determine (adjusted) amount of fragmented cRNA (x) needed for gel
analysis (at least 1 ug) plus hybridization cocktail:
Array type
Micro/Mini
Midi
Standard
Fragmented cRNA (for one probe array)
5 ug
10 ug
15 ug
2. Calculate amount of RNase-free water and 5X Fragmentation buffer
needed to have a final cRNA concentration between 0.5 ug/ul and 2 ug/ul:
a. Determine final volume (z) of the fragmentation reaction:
x ug cRNA to
x ug cRNA
2 ug/ul
0.5 ug/ul
b. 5X Fragmentation buffer should be 20% of final volume (2 ul per
8 ul RNA + water)
c. Final Fragmentation
x cRNA
5X Frag. buffer
RNase-free water
FINAL VOLUME
Reaction:
a ul (set by conc. of purified IVT product)
b ul (0.2z)
c ul (z – a – b)
z ul
3. Incubate fragmentation reaction at 94C for 35 min.
4. Chill on ice, then spin to collect condensation.
5. Save aliquot (at least 1 ug RNA) for gel electrophoresis. Store rest
of sample at –20C until ready to perform hybridization.
Gel Electrophoresis
1. Run aliquots of cDNA (optional), unpurified and purified cRNA, and
fragmented cRNA on a 1% agarose gel (or denaturing gel) to estimate yield
and size distribution of labeled transcripts.
2. Put 0.3 g agarose in 30 ml 1X TBE and heat until boiling.
3. Cool on shaker table until touchable, then add 1 ul ethidium bromide.
4. Pour gel and let sit until hard.
5. Mix RNA (samples or ladder) with 6X loading dye and heat to 65C for 5
min before loading.
6. Run at 105 V for 30 – 60 min.
7. Fragmented cRNA should be distributed between 35 and 200 bp.
Comparing unpurified to purified IVT product can help determine the
amount of loss during clean up, if similar amounts or proportions of cRNA
are loaded.
Preparing Hybridization Target
1. Mix according to Table 5-1 in Affy manual:
Fragmented cRNA
Control B2 (Thaw 65C for 5 min)
20X Hybridization Controls (Thaw 65C for 5 min)
Herring Sperm
BSA
2X Hybridization Buffer
Water
2. Equilibrate probe array to room temp.
3. Heat Hyb. cocktail to 99C 5 min. in heat block
4. Fill array with 1X hyb buffer and incubate at 45C in oven with
rotation for
10 min.
5. Transfer hyb cocktail to oven for 5 min at 45C.
6. Spin hyb cocktail at max. speed in centrifuge 5 min.
7. Remove buffer from array and replace with appropriate volume of
clarified hyb cocktail. Avoid any solid material on bottom of tube.
8. Hybridize 16 hours at 45C and 60rpm.
Affymetrix Protocol Day 4
End of Hybridization
9. Remove hyb. cocktail. Store on ice or in -20 for long-term storage.
Fill probe array completely with non-stringent buffer. Probe array can
now be stored for 3 hours at 4C.
Preparation of SAPE (Streptavidin Phycoerythrin) and Antibody (if
required)
SAPE (single stain) for Arrays with 50 um spots
For each array:
2x MES Buffer (stored at 4C)
50 mg/ml BSA (Affy box -20)
1 mg/ml SAPE (stored at 4C)
DI water
TOTAL
300 ul
24 ul
6 ul
270 ul
600 ul
SAPE (Antibody Amplification) for arrays with 24 um spots
For each array:
2X MES stain buffer
50 mg/ml BSA
1 mg/ml SAPE
DI water
TOTAL
600 ul
48 ul
12 ul
540 ul
1200 ul
Mix and divide into 2 aliquots of 600 ul each. These will be used for
stain 1 and 3.
Store in foil on ice.
Antibody Solution
For each array:
2X MES stain buffer
50 mg/ml BSA
10 mg/ml normal goat IgG
300 ul
24 ul
6 ul
0.5 mg/ml biotinylated antibody
DI water
TOTAL
3.6 ul
266.4 ul
600 ul
Run Fluidics Station for Staining and Washing
Scan Chip and Analyze
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