MicroArray Analysis of Total RNA (Cell line)
Materials:
Item
Vendor Catalog Number
353109
T75 Cell Culture Flask Fisher
14280036
PBS
Invitrogen
15596-026
Trizol Reagent
Invitrogen
14-959-1B
14mL Falcon Tube
Fisher
BP1145-1
Chloroform
Sigma
A416-4
Isopropanol
Sigma
14-959-49A
50mL Conical Tube
Fisher
111HPLC200CS4L
Ethanol
Sigma
15510-027
Agarose
Invitrogen
BP308-100
MOPS Buffer
Fisher
74104
Rneasy Mini Kit
Qiagen
Erie Scientific
22IX25-2-4635
LifterSlip - Small
Erie Scientific
25x60I-2-4789
LifterSlip - Large
Item
CyScribe FS cDNA Kit
Auto Seq G-50 Column
2.5 M NaOH
2 M HEPES free Acid
20X SSC
SDS 10%
Formamide
BSA
Hybrid Solution #1
0.5 mL microtube
Hybridization Cassette
Microarray Slide
Vendor
APBiotech
APBiotech
Sigma
Sigma
Fisher
Fisher
Sigma
Sigma
Ambion
Catalog Number
RPN6202
27-5340-01
BP359-212
H3375
BW16-009Y
BW16-007B
BP227-500
B-4287
8861
LS-9350-B
Life Science Prods.
Corning
UMCCC
2551
Equipment:
Item
Sorvall RC SC Plus
Centrifuge Rotor
Microcentrifuge
Spectrophotometer
Hybridization Oven
Vendor
Sorvall
Sorvall
Any
Any
Any
Catalog Number
SS-34
Item
Vacuum Concentrator
WaterBath - 42C
WaterBath - 68C
Thermocycler
Vendor
Labconco
Any
Any
Any
Catalog Number
Methods:
RNA Isolation: Trizol
-
Cells are grown to 60-70% confluent in T75 flask.
Aspirate media, wash with PBS, aspirate.
Add 7.5 mL of Trizol Reagent to flask, scrape lysed cells using cell scraper (if necessary).
Transfer reagent to 14 mL Falcon tube.
Trizol may be stored at -80 until used (up to 1 month).
-
If frozen, thaw to RT before use, otherwise, let sit at RT ~5min.
Add 1.5 mL of chloroform, mix vigorously.
Incubate at RT for 3 min.
Centrifuge at <12000 x g for 20 min., 4C (9000rpm in Sorvall RC SC Plus, SS-34 Rotor).
To a new 14 mL tube, transfer the clear (top) aqueous liquid from the tube containing Trizol. Do
NOT transfer any white precipitate!
Add 4 mL cold isopropanol, mix gently, and incubate for 10 min at RT.
Centrifuge at <12000 x g for 20 min at 4C.
Carefully pour off supernatant, leaving behind clear pellet.
Add 2 mL of cold 75% ethanol.
Centrifuge at <12000 x g for 5 min at 4c.
Carefully pour off supernatant, leaving behind clear pellet.
-
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-
Centrifuge at < 12000 x g for 1 min.
Remove remaining supernatant, allow pellet to air dry 10 min.
Resuspend RNA pellet by adding 50 uL DEPC-ddH20 (RNase free water).
Store at -80C
RNA Quantification and Quality
-
Using a 1:50 dilution of RNA in RNase free water, measure the absorbance of 260nm light waves
using a spectrophotometer.
Cell line RNA concentration should ideally be 1ug/ul or greater
Cell line RNA yield should ideally be 50ug/flask or better.
Cell line RNA ratio (A260/A280) should ideally be >1.8 (>1.6 is also acceptable).
Verify RNA quality by running a non-denaturing Agarose gel in 1X MOPS buffer using 1-3ug
total RNA.
Total RNA purification: RNeasy Kit
-
Follow kit protocol and directions (no deviations used; however, optional steps are omitted).
If Total RNA yield is > 100 ug use the RNeasy MIDI kit.
If Total RNA yield is < 100 ug use the RNeasy MINI kit.
You should expect to lose ~30-40% of Total RNA using this kit. (tRNA and other small RNA
molecules are excluded from purified RNA using this protocol).
Labeled cDNA Probe Generation: CyScribe First-Strand cDNA Labeling Kit
Denaturing and Annealling
- Centrifuge under vaccum (SpeedVac) 20 ug RNA to a volume of 9ul in a separate 0.5mL tube for
each probe (one Cy3-labeled and one Cy5-labeled).
- Add 1ul of oligo (dT) primer and 1ul of random nanomers. (Note: to enhance for mRNA
selection, you may change the amounts of primes to 1.5ul and 0.5ul, respectively.)
- Mix and spin down.
- Incubate the RNA/primer mixture at 70C for 5 min, then cool at RT for 10 min.
Labeling Reaction – To each tube…
- Add 4uL of 5x CyScript buffer.
- Add 2uL of 0.1 M DTT.
- Add 1uL dCTP nucleotide mix.
- Add 1uL CyScript reverse transcriptase.
- Then add the light sensitive CyDye-dCTP to the appropriate tubes (one Cy3, and one Cy 5).
- Mix, spin down, and then incubate at 42C for 1.5 hours.
Degradation of RNA – To each tube…
- Add 2uL of 2.5 M NaOH and incubate at 37C for 15 min.
- Add 10 uL of 2 M HEPES free acid, mix.
Purification of CyDye-labeled cDNA
- Prepare AutoSeq G-50 column as indicated by manufacturer.
- Add each labeled cDNA to separate columns.
- Collect flow-thru ensuring that each appears to have color (a red color for Cy3, and blue for Cy5).
- Top of column will also retain color (unincorperated CyNucleotides).
- Combine both Cylabeled cDNA probes in same 0.5 mL tube.
- Centrifuge under vacuum the mixture to ~5uL, should appear purple in color.
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Slide Prehybridization
-
Incubate microarray slide for 45 min. at 42C using 60mL of 5X SSC, 0.1% SDS, 25%
Formamide, 1% BSA in a coplin jar.
Wash slide with ddH20 (do not use Millipore water!!!)
Dry slide
Hybridization
-
-
Preheat slide and cassette to 68C
Preheat Hybridization solution to 68C
Add sufficient Hybrid. Sol. to cover slide to the cDNA mixture.
o 25uL when using plastic coverslip and small (30) lifterslip.
o 50uL when using medium (40) lifterslip.
o 75uL when using large (50) lifterslip.
Incubate Hybrid. Solution with cDNA mixture at 68C 5 min.
Add solution to microarray surface (try to avoid placing directly on spot constellations) or at the
top edge of the lifterslip.
Add plastic coverslip (no air trapped underneath) if not using lifterslip.
Add 10uL water to cassette, wrap assembled cassette in foil, and place at 45C overnight.
Washes
1.
2.
3.
4.
5.
6.
2X SSC + 0.2% SDS, rotate 45C for 10 min.
2X SSC, rotate RT for 10 min.
0.2X SSC, rotate RT for 10 min.
Rinse slide.
Dry.
Scan immediately.
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