Using Genisphere 3DNA Submicro Kit
General Instructions
Indirect Labeling (Dendrimer Approach) [link ppt file]
The following protocol is an abbreviated version of the protocol that accompanies the kit. Read through the kit protocol before following the steps below.
- Use only RNase/DNase free reagents/supplies. Wear gloves at all times
- Cyanine 3/Cyanine 5 dyes and reagents containing these dyes are light sensitive. Care should be taken to minimize exposure of these reagents to light
Determination of Labels and Amount of RNA Needed
- The untreated/control sample should be labeled with Cyanine 3 (Cy3)
- The treated sample should be labeled with Cyanine 5 (Cy5)
- 3-5 µg of total RNA is required per reaction
cDNA Synthesis
- In a 1.5-mL microfuge tube, combine “x” µL of 3-5 µg total RNA and 3 µL RT primer (Vial 2) (Cy3 or Cy5). Add RNase free water to a final volume of 10 µL. (This is the RNA-RT primer mix.)
- Heat to 80°C for 10 minutes. Cool on ice
- Add 1 µL of RNase inhibitor (RNasin, Promega, Catalog No. N2511)
- In a separate microcentrifuge tube, combine 4 µL of 5X RT Buffer (Vial 5), 1 µL of dNTP mix (Vial 4), 4 µL of RNase free water and 1 µL of RT enzyme (Vial 3). (This is the reaction mix.)
- Add 10 µL of reaction mix to the 11 µL of RNA-RT primer mix
- Mix and incubate at 42°C for 2 hours
- Add 3.5 µL of 0.5 M NaOH/50 mM EDTA
- Incubate at 65°C for 10-15 minutes
- Add 5 µL of 1 M Tris-CL, pH 7.5
- For dual channel expression, transfer Cy3 cDNA mixture into tube containing Cy5 cDNA mixture. Rinse Cy3 tube with 10 µL of 10 mM Tris, pH 8.0, 1 mM EDTA. Combine wash with the cDNA mixtures
- Add 1 µL of 20 mg/mL glycogen
- Add 175 µL of 3M ammonium acetate and mix
- Add 625 µL of 100% ethanol
- Incubate at -20°C for 30 minutes
- Centrifuge at maximum speed at 4°C for 15 minutes
- Aspirate supernatant. Add 300 µL of 70% ethanol
- Centrifuge at maximum speed at 4°C for 5 minutes
- Spin dry pellet in speed vac for 5-10 minutes at room temperature
Prehybridization
- Prehybridize slide in 50-mL conical tube, containing 5X SSC, 0.1% SDS and 1% BSA (Fraction V), at 50°C for 30 minutes
- Wash slide in 2X SSC at room temperature for 3 minutes with rocking
- Wash slide in 0.2X SSC at room temperature for 3 minutes with rocking
- Spin dry. Slide is ready for hybridization now
Hybridization
- Thaw and resuspend hybridization buffer (Vial 6) by heating to 65°C for 10 minutes and mixing occasionally
- Add 1 µL of Anti-Fade reagent (Vial 8) to every 100 µL of hybridization buffer. Add 100 ng of Cot-1 DNA for every microgram of total RNA to every 100 µL of hybridization buffer. Heat to 90°C for 5 minutes
- Resuspend pellet (see step 18 in cDNA Synthesis) in hybridization buffer made accordingly:
10 µL Rnase free water
22 µL hybridization buffer (from step above)
2.5 µL Cy3-3DNA capture reagent (Vial 1)
2.5 µL Cy5-3DNA capture reagent (Vial 1)
2.0 µL Oligo dT Blocking reagent (Vial 9)
1.0 µL High-End Differential Enhancer (Vial 10)
40 µL total - Incubate hybridization mix at 55-60°C for 15-20 minutes.
- Add to array. Cover with coverslip. Avoid air bubbles. Hybridize overnight at 60°C.
Washes
- Wash slide at 60°C for 15 minutes in 2X SSC, 0.2% SDS.
- Wash slide for 15 minutes at room temperature with 2X SSC with rocking.
- Wash slide for 15 minutes at room temperature with 0.2X SSC with rocking.
- Spin dry and scan slide.