• CamSysBiol
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Klenow labelling of double stranded DNA derived from 3 to 5 µg total RNA

Outline

Klenow labelling of double stranded DNA can be used when the amount of biological material is limiting. With this method, amplification by in vitro transcription of the sample can be avoided. The RNA samples are reverse transcribed to cDNA and second strand synthesis is then performed to obtain double stranded DNA (dsDNA). Fluorescent dyes are incorporated using Klenow fragment and the labelled samples are then subjected to paired competitive hybridisations.

Equipment and reagents

• Oligo(dT)23 anchored primer (Sigma; Cat. No. 04387)
• Superscript III Reverse Transcriptase (Invitrogen; Cat. No. 18080-044)
• RNAsin (Promega; Cat. No. 18064-014)
• dATP, dCTP, dTTP and dGTP (Sigma; Cat. No. dNTP-100A)
• Second strand buffer (Invitrogen; Cat. No. 10812-014)
• DNA Polymerase I (Invitrogen; Cat. No. 1801-025)
• RNaseH (New England Biolabs; Cat. No. M0297S)
• E. coli DNA ligase (GE Healthcare Bio-Sciences AB; Cat. No. E70020Z)
• Phenol:Chloroform:Isoamylalcohol (Sigma; Cat. No. P2069)
• Phase Lock Gel (Helena Bioscience; Cat. No. 0032 007.953)
• AutoSeq G-50 column (GE Healthcare Bio-Sciences AB, Cat. No. 27-5340-01)
• Sodium Chloride (VWR; Cat. No. 102414P)
• Gene Elute LPA (Sigma; Cat. No. 56575)
• 100% Ethanol
• 70% Ethanol
• DEPC - Diethyl pyrocarbonate (Sigma; Cat. No. D 5758)
• DEPC-treated MilliQ water
• Bioprime DNA Labeling System (Invitrogen; Cat. No. 18094-011)
• Cy3 dCTP (GE Healthcare Bio-Sciences AB; Cat. No. PA 53021)
• Cy5 dCTP (GE Healthcare Bio-Sciences AB; Cat. No. PA 55021)
• Sonicated Salmon Sperm DNA (Invitrogen; Cat. No. 15632-011)
• Hettich micro 20 centrifuge
• Grant QBT2 hot-block
• Savant Speed Vac
• Dyad thermal cycler (PCR block)

Removal of RNase

All materials should be autoclaved and only handled using gloves. Glassware should be baked at 180 °C overnight. Water and solutions should be treated with DEPC. The work area can be cleaned using RNase Zap to further limit the risk of RNase contamination. If at all possible, it is also a good precaution to use a separate set of pipettes for RNA work.

Procedure

Preparing the dNTP mixes

Making the 10mM dNTP mix:

1. Make a large 10 mM dNTP mix for the RT-reaction and second strand synthesis
   • 100 µl 100mM dNTA
   • 100 µl 100mM dNTT
   • 100 µl 100mM dNTG
   • 100 µl 100mM dNTC
2. Then make up to 1 ml using DEPC-water
3. Aliquot the dNTP mix and then store at -20 °C

Making the 10 X low-C dNTP mix:

1. Make a large 10 X low-C dNTP mix for the labelling reaction (5 mM A-,G-,T-dNTPs and 3mM C-dNTP)
   • 25 µl 100mM dNTA
   • 25 µl 100mM dNTT
   • 25 µl 100mM dNTG
   • 15 µl 100mM dNTC
2. Then make up to 500 µl using DEPC-water
3. Aliquot the 10 X low-C dNTP mix and then store at -20 °C

Reverse Transcription reaction:

The following steps are performed in 200 µl PCR tubes and the PCR block

1. Take between 3 to 5 µg of the extracted RNA (small scale RNA extraction protocol), add 1 µl of Oligo(dT)23 anchored primer (500 ng/µl) and then make up to a total volume of 5 µl with DEPC- water
2. Incubate at 65 °C for 10 min
3. Snap freeze on ice
4. Make up a premix for the RT reaction:
   • 4 µl Superscript Buffer (from superscript kit)
   • 2 µl 0.1M DTT (from superscript kit)
   • 1 µl 10 mM dNTP mix
   • 0.25 µl 40 U/µl RNasin
   • 0.75 µl 200 U/µl Superscript III
5. Add 8 µl to each sample
6. Incubate at 46 °C for 2 hours
7. Incubate at 65 °C for 15 minutes
8. Snap freeze on ice

Second strand synthesis:

The following steps are performed in 200 µl PCR tubes and the PCR block

9. Make up a premix for the second strand synthesis:
   • 9.15 µl DEPC-water
   • 7.5 µl Second Strand Buffer
   • 0.75 µl 10 mM dNTP mix
   • 2 µl 10 U/µl DNA Polymerase I
   • 0.1 µl 5 U/µl RNaseH
   • 0.5 µl 10 U/µl E. coli Ligase
10. Add 20 µl to each sample
11. Incubate at 16 °C for 2 hours

Purification of dsDNA:

The following steps are performed in 1.5 ml microfuge tubes

12. Make the sample up to 100 µl (add 60 µl DEPC-water)
13. Immediately before use pellet Phase Lock Gel (PLG) tube at 13,000 rpm for 30 seconds
14. Add the cDNA and an equal volume (100 µl) of (Phenol/Chloroform/Isoamylalcohol pH 8.0) to the PLG tube and shake for 15 seconds (do not vortex)
15. Centrifuge for 5 minutes at 13000 rpm
16. Transfer upper phase to a new 1.5 ml microfuge tube
17. Reduce volume of sample to approximately 30 µl, by placing in a speed vac with medium heat
18. Prepare the G50 columns:
    • Resuspend the resin in the G-50 column by vortexing gently
    • Loosen the cap a quarter turn and snap off the bottom closure
    • Place the column in a 1.5 ml tube
    • Pre-spin column at 5,000 rpm (2000 x g) for 1 min to remove the buffer
    • Remove the top cap and place column in a new 1.5 ml tube
19. Pipette the sample to the G50 columns onto the centre of the angled surface of the compacted resin bed being careful not to disturb the resin. Do not allow any of the sample to flow around the sides of the bed.
20. Centrifuge for 1 minute at 5000 rpm, discard the column
21. Adjust the volume to 100 µl with DEPC-water
22. Precipitate the dsDNA:
    • 3.5 µl 5M Sodium Chloride
    • 0.5 µl LPA
    • 220 µl 100% Ethanol
23. Incubate for 2 hours at -20 °C (or 15 minutes at -80 °C)
24. Centrifuge for 30 minutes at 13000 rpm
25. Remove supernatant and wash pellet with 70% Ethanol, centrifuge for 2-3 minutes
26. Dry pellet and dissolve in 10 to 20 µl DEPC-H2O
27. Measure concentration on Nanodrop

Klenow labelling:

The following steps are performed in 200 µl PCR tubes and the PCR block

28. Take up to 1 µg double stranded DNA and make up to a total volume of 13 µl with DEPC-water
29. Add 10 µl 2.5x Random Primer Reaction Buffer (supplied in the Bioprime Labelling System Kit)
30. Incubate at 100 °C for 5 minutes
31. Snap freeze on ice
32. Mix together the following to make a master mix:
    • 0.5 µl 10 X low-C dNTP mix
    • 1 µl Cy3 or Cy5 dCTP
    • 0.5 µl 40U/µl Klenow
33. Add 2 µl to each sample and mix by pipetting up and down
34. Incubate at 37°C for 2 to 3 hours
35. Stop the reaction by adding 2.5 µl Stop Buffer (supplied in the Bioprime Labelling System Kit)
36. Combine the Cy3 and Cy5 pairs

Probe clean-up:

It is important to separate the fluorescently-labelled probe from any unincorporated dye and nucleotides. AutoSeq G-50 columns are quick and easy to use. Microcon 30 columns (Millipore) or Qiaquick PCR purification columns (Qiagen) work equally well.

Purify probe using an AutoSeq G-50 column as follows:

37. Prepare the G50 columns (need 2 columns per combined sample):
    • Resuspend the resin in the G-50 column by vortexing gently
    • Loosen the cap a quarter turn and snap off the bottom closure
    • Place the column in a 1.5 ml tube
    • Pre-spin column at 5,000 rpm (2000 x g) for 1 min to remove the buffer
    • Remove the top cap and place column in a new 1.5 ml tube
38. Pipette half the sample to the G50 columns onto the centre of the angled surface of the compacted resin bed being careful not to disturb the resin. Do not allow any of the sample to flow around the sides of the bed.
39. Centrifuge for 1 minute at 5000 rpm
40. Place a second column into the same 1.5 ml microfuge tube and then add the second half of the sample. Spin for 1 minute and 5,000 rmp.
41. Reduce volume of sample to between 2 to 5 µl by placing in a speed vac with medium heat
42. Add 2 µl of sonicated salmon sperm DNA

The samples have now been labelled and combined together for hybridisation to a microarray with the blocking agent, sonicated salmon sperm DNA. This material should now be used immediately to prevent any decay. Please refer to the appropriate hybridisation protocol for the next steps.

R. Auburn (21-08-2006).