ORNL MICROARRAY HYBRIDIZATION PROTOCOLS
互联网
Direct labeling of total RNA with Cy3 and Cy5:
RNeasy® Mini Kit (Qiagen; Cat # 74106)B. REAGENT PREPARATION
SuperScript II RT (200U/µL) (Life Technologies; Cat # 18064-014)
QIAquick PCR Purification Kit (Qiagen; Cat # 28106)
100 mM dNTP Set PCR grade (Life Technologies; Cat # 10297-018)
Primers: Poly d(T) [20-mers] 2.5 mg/mL (avoids labeling the gDNA and rRNA.)
Anchored poly d(T) primers
(Archaic) Random Hexamer primers (3mg/mL) (Life Technologies; Cat # 48190-011)
Cyanine 3-dUTP # NEL578 or Cyanine 5-dUTP #NEL579 (Perkin Elmer/NEN)
| 50X [ ] | Stock [ ] | For 1.0 ml of 50X use: | |
| dTTP | 5 mM | 100mM | 50 µL |
| dATP | 25mM | 100mM | 250 µL |
| dGTP | 25mM | 100mM | 250 µL |
| dCTP | 25mM | 100mM | 250 µL |
| RF-ddH20 | 200 µL |
| Master Mix: | (Per Reaction) | USED TODAY: |
| 5X Enzyme Buffer | 6 µL | |
| 0.1 M DTT | 3 µL | |
| 50X dNTPs | 0.6 µL | |
| Superscript II RT | 2 µL |
C. PROCEDURE: This protocol uses 30-100µg of total RNA (Prepare two 30-100 µg samples for comparative hybridizations using two-colors). To use the volumes listed, the RNA must be at ? 2.5 mg/ml. Protect slide from light at all times until imaged to prevent bleaching of dyes.
Secure the top of the microfuge tube with a clip, and heat to 95°C for 5 min to denature.
Centrifuge 2 minutes. Do not transfer to ice.
Apply targets to the microarray, avoiding bubbles. Place coverslip slowly down onto array. If bubbles form, do not attempt to "push" them out. This will damage your array.
Hybridize the array in a humidified container overnight (16-18 hours) in a 42°C water bath.
(floating is usually OK, but can be placed on a support to keep it level)
After hybridization, remove slide from Hybridization chamber (avoid water dripping onto array).
Protect slide from light at all times until imaged to prevent bleaching of dyes.
Indirect labeling of RNA using aminoallyl-modified nucletides and monoester reactive Cy-dyes
1. MATERIALS
2. REAGENT PREPARATION
3. PROCEDURE
Direct labeling of small amounts of total RNA
Sub-micro Cy3&Cy5 Labeling of RNA for Microarrays #2
Modified to use Genisphere Sub-micro kit
Mix and zing.
Heat to 80°C, 10min, then immediately ice
In separate mfuge tube on ice mix: ("Reaction Mix" 10ul)
4ul 5X RT buffer (Vial 5)
1ul dNTP mix (Vial 4)
4ul RF H20
1ul (200 Units) reverse transcriptase enzyme (Vial 3)
Gently mix and zing.
Add primer mix to reaction mix (final volume 20ul) [primer mix now actually about 7ul]
Mix and incubate at 42°C for 2 hours
Stop reaction with 3.5ul of 0.5M NaOH, 50mM EDTA [Probably should make fresh]
Incubate at 65°C for 10min
Immediately neutralize the mix with 5ml of 1M Tris-HCl, pH= 7.5
・ If only using single label, add 38.5ml of 10mM Tris-HCl, pH=8.0, 1mM EDTA (Volume = 67ml)
・ For dual labels, combine the Cy3 with the Cy5 (Volume = 57ul). Then rinse Cy3 tube with 10ul of 10mM Tris-pH=8, 1mM EDTA, and combine with label. (Volume = 67ul)
Add the 0.1 ul of 1mg/ml C0T-1 DNA (Volume = 67ul)
Genisphere recommends 20 ug of Linear Polyacrylamide (LPA) (2ul of 10ug/ul) for carrier. (Volume = 69ul)
~undefined************~Kbr_~H~M~2~1~0Concentration_via_Precipitation~I~Kbr_~H~M~2~1~0Add_2_volumes_3M_ammonium_acetate_and_mix_~A140ul~B~Kbr_~H~M~2~1~0Add_2.5_volumes_of_100~7_EtOH~Kbr_~H~M~2~1~0Incubate_at_~F20°C 30 minutes
Centrifuge 15 minutes
Discard supe, and wash pellet with 70% EtOH
Centrifuge 5 minutes, discard supe
Dry in Speed-Vac just to the point of pellet (Do not dry completely or it'll be hard to resuspend)
~undefined************~Kbr_~H~M~2~1~0Thaw_DNA_Hyb_buffer_~AVial_6~B_by_heating_to_65°C with inversion (not vortexing?)
Thaw 3DNA capture reagent (Vial 1) by hand and vortex thoroughly to resuspend aggregates
Resuspend cDNA Samples in 5ml sterile water
Add 2.5ul each of Cy3 and Cy5 3DNA Capture reagents (Vials 1) to cDNA samples
If using Single channel, use only the one Capture reagent
Add 10ul Hybridization Buffer (Vial 6)
Incubate at 65°C for 15-20 minutes
Add probe mix to microarray and Hyb O/N at 55-65°C.
Washes:
10'@55°C using 2XSSC, 0.2% SDS
10'@RT° using 2XSSC
10'@RT° using 0.2XSSC
Image Arrays
GENERAL PREHYBRIDIZATION AND HYBRIDIZATION PROTOCOL
Protecting Cy-reactions from light
ORNL Aminosilane Slide spotting protocols
Postprocessing
ORNL Pre-hybridization station
ORNL Hybridization Chamber (Made from a tip box!)
ORNL High-Throughput Wash Station (Made from histological staining setup)
