Cancer Genomics Experimental Protocol

Overview

Total RNA (mRNA)
| Approx. 5 hours
v
dscDNA
| Approx. 8 hours
v
In Vitro Transcription
| Approx. 16 hours
v
Hybridization
| Approx. 1 hour
v
Wash/Stain/Wash/Scan

First strand cDNA synthesis


  1. Add 10 uL total RNA (20 ug) in DEPC H2O
    1 uL 100 pmol/ul T7-(T)24 primer (GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24)
  2. Mix (quick spin if needed)
  3. Heat @ 70 C, 10 min
  4. Put in ice bucket
  5. Add on ice to RNA/primer mix:
  6. Heat @ 37, 2 min
  7. Add 2 uL SSII RT (400 U total)
  8. Mix (quick spin if needed)
  9. Heat @ 42 C, 1 hour
  10. Proceed to "Second strand cDNA synthesis"

Second strand cDNA synthesis

  1. Ice all reagents and 1st strand tubes
  2. Add to 1st strand tubes:
  3. Mix (quick spin if needed)
  4. Incubate @ 16°C, 2 hours
  5. Store @ -80 C


Clean-up of dscDNA

  1. Spin Phase-Lock tubes @ max, 30 sec
  2. Add all of the cDNA reaction (approx. 150 uL)
  3. Add equal volume buffer saturated phenol (or phenol/chloroform)
  4. Vortex lightly
  5. Spin @ max, 2 min
  6. Transfer upper phase to new tube
  7. Add
  8. Mix
  9. Spin @ max, R.T., 20 min
  10. Decant supernatant (watch for pellet)
  11. Wash pellet twice with 80% EtOH
  12. Speed vacuum to dry
  13. Resuspend in 1.5 uL DEPC H2O

In Vitro Transcription

  1. Thaw and room temperature all reagents
  2. Make NTP mix (per tube):
  3. Add to cleaned dscDNA tube:
  4. Mix (quick spin if needed)
  5. Incubate @ 37 C, 6 hours

IVT Clean-up

  1. Add to IVT reaction tube:
  2. Mix
  3. Add 250 uL 100% EtOH
  4. Transfer sample to RNeasy spin column
  5. Spin @ max, 15 sec
  6. Transfer spin column to new collection tube
  7. Add 500 uL RPE buffer
  8. Spin @ max, 15 sec
  9. Transfer spin column to new collection tube
  10. Add 500 uL RPE buffer
  11. Spin @ max, 2 min
  12. Transfer spin column to new collection tube
  13. Add 50 uL DEPC H2O to membrane of spin column
  14. Let soak for 4 min
  15. Spin @ max, 1 min
  16. Repeat 13-15 using 1st elution as the 2nd elution
  17. Take OD (1:50 dilution)
  18. Run on a 1% agarose gel using denaturing sample buffer (See Appendix A)

Fragmentation of cRNA

  1. Add to separate tube:
  2. Mix
  3. Heat @ 95, 35 min
  4. Add:
  5. Adjust volume with DEPC H2O to 900 uL total volume
  6. Store @ -80 C

As per maufacturer's protocols:

Information about DNA Chips and Equipment



Appendix

Gel using Denaturing Sample Buffer


1. Make Sample Buffer:

2. Add 10 uL Sample Buffer to each sample and controls to be run

3. Heat @ 65 C, 10 min

4. Run on 1% Agarose gel