Protocol cDNA-Microarray Hybridization
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RNA preparation
We recommend the following protocols for extracting total RNA:
cultured cells: Quiagen RNeasy Kit
Trizol (Gibco)
tissues: Quiagen RNeasy Kit
modified LiCl extraction protocol
(Auffray and Rougeon, Eur. J. Biochem. 107:303-314, 1980)
RNA precipitation (total RNA 100 - 150 µg)
Probe preparation
- 1/10 vol 3M NaOAc (ph 5.2)
- 2.5 vol 95% ethanol
- mix
- store at -70 °C for 20 min (or -20 °C for 1 h or O/N)
- spin at max. rpm for15'
- discard supernatent
- add 1x vol 75% ethanol
- discard supernatent
- air dry pellet (make sure ALL ethanol is evaporated)
- resuspend in 17 µl DEPC-H2O
- place on ice
-
Anneal RNA
- start with 17 µl of resuspended RNA per probe
- Add 2 µl oligo dT (12-18)
- Final Volume: 19 µl (per tube)
- incubate for 3-5 min at 65 °C
- place on ice
- Make fluorescent cDNA probe
-
- 8 µl 5x first strand buffer
- 4 µl 0.1m DTT
- 4 µl 10x low dT dNTP
- 4 µl Cy3 or Cy5 dUTP
- 1 µl RNAsin
- Final Volume: 21 µl (per tube)
- Add annealed RNA to flourescent mix
- Final Volume: 40 µl (per tube)
- Add 2 µl Superscript-RT (Gibco)
- Incubate at 42 °C for one hour
- add 2 µl Superscript-RT
- Incubate at 42 °C for another hour
- Heat at 94 °C for 2-3 min
- Clean cDNA from RNA
-
- Add 44 µl H2O to each tube
- 10 µl 10x RNAse one buffer
- 2 µl RNAse one
- Incubate at 37 °C for 10 min
- Heat at 94 °C for 2-3 min
- Combine both probes and add 30 µl of Strata Clean Resin
- Mix and incubate RT for 1-2 min, pellet resin by brief centrifugation, save supernate.
- Wash pellet with 100 µl of water, pellet and combine supernatents
- Concentrate probes
-
- Add to micron YM 50 column
- Add 200 µl H2O
- Mark tube & column for orientation
- Centrifuge at RT for 8-10' at 10,000-12,500g
- Wash 3x with 400 µl H2O; discard flow through
- Invert column, place into new tube and spin 15 sec at 1000g to collect probe
- Check volume and adjust to 19.5 µl
- Prehybridization of probe
-
- Add 40.5 µl hybridization solution to19.5 ul probe
- add 1µl blocking solution
- total 60ul
- Heat at 94 °C for 1 min
- Centrifuge at 13,000 rpm for 5' and transfer supernatant to clean tube
- Prehybridize probe at 50 °C for one hour
- Scan
Solutions
Filter all solutions that come in contact with slide except blocking solution
low dTTP/dNTP solution
25 µl dGTP
25 µl dATP
25 µl dCTP
10 µl dTTP
415 µl DEPC H20
500 µl total
Prehybridization solution (Available from facility)
3.5 ml formamide
2 ml 20x SSPE
0.5 ml 10% SDS
0.5 ml 50x Denhardt's
0.2 ml ss salmon sperm DNA
3.3 ml ddH20
10 ml total
Hybridization solution (Available from facility)
700 µl formamide (35%)
50 µl 20% SDS (0.5%)
100 µl 50x Denhardt's (2.5x)
400 µl 20x SSPE (4x)
1.25 ml total
Blocking solution (20x stock)
40 µl polydA (1ug/ul)
8 µl tRNA (10ug/ul)
200 µl human/mouse Cot1 DNA (1ug/ul)
240 µl total
ethanol precipitate the combined reagents
Resuspend in 20 µl filtered ddH2O