Protocol:

1. Observe membrane and note any abnormalities about growth after spotting. Check pencil numbers and robot puncture marks.

2. Handle membrane with 2 forceps holding it for diagonal corners.

3. Place on Whatman pre-wetted in Denaturing Solution. Leave for 4 minutes.

4. Place membrane on fresh Whatman pre-wetted in Denaturing Solution and transfer to glass plate sitting on empty pipette tip box in boiling waterbath. Leave for 4 minutes with covered lid.

5. Place membrane on Whatman pre-wetted in Neutratlizing Solution. Leave for 4 minutes.

6. Place membrane on dry Whatman. Leave for 1 minute.

7. Submerge membrane under a 600ml pre-warmed (37 °C) PROPK Solution containing ~150mg/ml fresh Proteinase K. Do NOT shake. Leave in incubator for 30-50 minutes.

8. Place membrane on dry Whatman.

9. Cover with a NUNC. Leave overnight.

10. UV cross-link.

11. Store between dry Whatmans.

Denaturation

1. Prepare the following:

25 µg total RNA/ 1µg poly A⁺ RNA x µl

1 µg oligo dT₁₉V (2 µg/µl) 0.5 µl

8 µg oligo dT₃₀ (8µg/µl) 1.0 µl

GFP poly A⁺ RNA (10ng/µl) 0.5 µl

ddH₂O x µl

Final volume 10.5 µl

2. Heat at 70 °C for 10 minutes, cool to 42 °C, slowly.

Note: Heat in 70 °C heat block then take out block and allow to cool on bench top until temp is 42 °C.

Reverse Transcription

3. To sample from above add the following:

dNTP mix (800 µM dATP/dGTP/dTTP, 5 µM dCTP) 1.0 µl

5x First Strand Buffer 5.0 µl

10x DTT 2.5 µl

[α-³²P]-dCTP (10µCi/µl) 5.0 µl

SuperScript II (200 U/µl) 1.0 µl

Final volume 25.0 µl

4. Incubate at 42 °C for 1 hour.

Hydrolysis of RNA

5. Sequentially add the following:

1% SDS 1.0 µl

500 mM EDTA 1.0 µl

2 N NaOH 3.0 µl

6. Heat at 65 °C for 5 minutes

7. Then add the following:

1 M Tris.HCl pH 7.6 10.0 µl

2 N HCl 3.0 µl

8. Incubate at RT for 10 minutes.

9. Add ddH₂O 7.0 µl

Final volume 50.0 µl

10. Purify probe over G-50 Micro Column (Pre-spin col umn at 3500 rpm for 1 minute, apply probe to column, spin at 3500 rpm for 2 minutes.)

Note: Save 1µl for counting and 1µl for denaturing gel.

Prehyb membrane

11. Incubate filter at 65 °C for 6-20 hrs in 10 ml Hybridization solution (5x SSC, 5x Denhardt�s, 0.5% SDS, 100 µg/ml ssDNA)

5ml 10ml 15ml 20ml 25ml 30ml 50ml

20x SSC 1.25 2.50 3.75 5.00 6.25 7.50 12.5

50x Denhardt�s 0.50 1.00 1.50 2.00 2.50 3.00 5.00

20% SDS 0.13 0.25 0.38 0.50 0.63 0.75 1.25

ssDNA 0.05 0.10 0.15 0.20 0.25 0.30 0.50

ddH₂O 3.10 6.20 9.20 12.3 15.4 18.5 30.8

Probe preparation

12. Boil probe 5 minutes, place on ice at least 5 minutes.

13. Add 2 µg poly A₇₂ to 2 ml Hybridization solution.

14. Add denatured probe to the 2 ml Hybridization solution.

15. Incubate 1 hr at 65 °C.

16. Add 3 ml Hybridization solution.

Hyb membrane

17. Incubate filter in 5 ml Hybridization solution for 20 hrs at 65 °C.

Washes

18. Wash membrane 5 minutes at RT in 2x SSC/0.1% SDS.

19. Wash membrane 20 minutes at 65 °C in 2x SSC/0.1% SDS.

20. Wash membrane 1 hour at 65 °C in 0.1x SSC/0.1% SDS.

21 (Optional). If needed, repeat 1 hour at 65 °C 0.1x SSC/0.1% SDS.

Adapted from Huntsman Cancer Institute Katze Lab Protocols

2. Prepare 0.3 to 1 pmole of each Cy³ or Cy⁵ labeled primer in 50µl 1X hybridization solution (50% formamide, 5X SSC, 0.1% SDS, 100µg/ml salmon sperm DNA).

3. Pipet 50µl hyb solution with probe onto long edge of slide.

4. Cover with long cover slip, avoid air bubbles (gives high background).

5. Incubate overnight (12hrs) at room temperature in humid chamber (moist paper towel in bottom of relatively airtight container).

6. To remove coverslip, submerge slide in 2X SSC/0.2% SDS.

Turn on scanner now if you want to scan immediately after processing, scanner needs at least 40 minutes to warm up.

7. Wash 5 min in 2X SSC/0.2% SDS at room temperature.

8. Wash10 min at room temp with 2X SSC/0.1% SDS.

9. Rinse slide 10 seconds in ddH₂0.

10. Dry with compressed air.

11. Store in dark until ready to scan.

12. Scan in Array Scanner II in both channels (make sure scanner has had at least 40 minutes to warm up).

Reagents:

• Forward Gene Specific Primer (IDT)
• Reverse Gene Specific Primer (IDT)
• Random decamer primer (from Ambion RetroScript Kit: 1710, $206.00/kit or Ambion, 5722G, $50.00/80µl)
• RNase Inhibitor (from Ambion RetroScript Kit: 1710, $206.00/kit)
• Reverse Transcriptase (M-MLV) (5x RT Buffer from Ambion RetroScript Kit: 1710, $206.00/kit)
• 10x RT Buffer, otherwise referred to as 10x first strand buffer (from Ambion RetroScript Kit: 1710, $206.00/kit)
• 10x PCR Reaction Buffer (from Ambion RetroScript Kit: 1710, $206.00/kit)
• 50 mM MgCl₂ (from Ambion RetroScript Kit: 1710, $206.00/kit)
• SuperTAQ DNA Polymerase (Ambion, 2050, $48.00/50 U or 2052, $190.00/250U)
• 18S rRNA PCR Primer Pair (Ambion kit, 1716 $155.00/kit)
• Control Template RNA (Ambion kit, 1716 $155.00/kit)

Protocol:

Reverse Transcription

1. Adjust concentration of RNA to 100 ng/µl (The RNA should be DNase treated or equivalent).

2. Set up two 500 µl tubes: mark one tube as (-)RT control – it will have no reverse transcriptase; mark the other with the sample name. Both tubes will will contain the same sample RNA.

3. For each tube mix together:

   RNA (100-450ng) 2 µl
   Ramdom decamer (in excess) 2 µl
   H₂O to equal total volume 8 µl
   Total volume 12 µl
   For a positive control use 2 µl of the RNA Control Template (Ambion).

4. Heat at 75°C for ten minutes; briefly centrifuge and keep on ice 30 seconds or until ready.

5. While heating above, prepare the RT Reaction Cocktail for the sample(s) and the controls (separate):

   Per Sample or (+)RT Control		Per (-)RT Control
   5x RT Buffer	2 µl	5x RT Buffer	2 µl
   2.5mM dNTP	4 µl	2.5mM dNTP	4 µl
   RNase Inhibitor (40 U/µl)	1 µl	RNase Inhibitor (40 U/µl)	1 µl
   M-MLV Reverse Transcriptase	1 µl	H2O	1 µl
   Total Volume	8 µl	Total Volume	8 µl

   Note: When doing multiple reactions, make up a master mix for all samples allowing 10% extra to permit for pipetting errors. Aliquot 8 µl/sample or control and add to the reaction mixture from step 2.

6. Add 8 µl of the RT Cocktail mix to the 12 µl of RNA mix. Mix by pipetting up and down, centrifuge briefly.

7. Incubate at 42°C for 1 hour.

8. Centrifuge briefly and store at -20°C until ready to continue.

Polymerase Chain Reaction

1. For linear range PCR, we find it best to use 10 cycle-increments plus controls.
   Controls are:
   • -a positive RT Control
   • -a negative PCR control to ensure no reagent contaminants
   • -a negative RT sample control (no M-MLV RT) to ensure the RNA used is free of DNA contaminants. The (+) RT and (-) PCR control can be use for an entire experiment, for all conditions.
     The (-) RT sample controls, however, are needed per condition being tested.

1. For each of the controls add 1 µl of the following. | | | | | — | ————————— | ——————————————– | | 1a. | Positive RT Control-Mock | 1µl cDNA from RNA Control Template (Ambion) | | 1b. | Negative PCR Control | 1µl H₂O | | 2a. | Negative RT Control-Mock | 1µl (-)RT Mock cDNA | | 2b. | Negative RT Control-Treated | 1µl (-)RT Treated cDNA |

For Linear Range

1. For each sample, add 1 µl cDNA for a total of 50 µl.
2. Distribute 50 µl each into 10 separate tubes with each representing a different cycle.
3. Set and run the following PCR cycles:
   1. 95°C for 3 minutes
      start cycles:
   2. 95°C for 45 seconds
   3. 58°C for 45 seconds
   4. 72°C for 45 seconds
      REPEAT from step “b” 35 times
      Note: Preheat thermocycler before placing the sample tubes in the machine.

1. Remove the appropriate tube after the designated cycle is over. Ideally you should run a final extension cycle in another thermocycler (10 minutes at 72°C). Put on ice for 1 minute. Keep at 4°C until ready to load into gel. Store PCR product at -20°C.
2. Add 2.5 µl of dye to 10 µl of sample. Run samples on a 2% agarose gel in 1x TAE buffer at 100 Volts until done or at 25 Volts overnight. Stain gel with Ethidium Bromide or Sybr Green.
3. To image on Phosphoimager (Storm): for 100 ml gel, prepare 250 ml 1x TAE buffer with 25 µl Sybr Green stain. (Stain the gel in 1:10,000 dilution Sybr Green stain in 1x TAE buffer.) Shake gently for at least a hour. Sybr Green can be reused.
   Note: Keep the staining solution in the dark by wrapping the container with aluminum foil.
4. Observe gel under UV light and photograph the image. Also, scan the gel under STORM fluorimager.
5. Determine the optimal range by observing the linear range of the gel. Take two points within the optimal range to continue on with the Quantitative RT PCR.

1. Observe gel under UV light and photograph the image. Also, scan the gel under STORM Fluorimager to be able to analyze quantitative differences.

Troubleshooting

Reagents:

• Forward Gene Specific Primer (IDT)
• Reverse Gene Specific Primer (IDT)
• Random decamer primer (from Ambion RetroScript Kit: 1710, $206.00/kit or Ambion, 5722G, $50.00/80µl)
• RNase Inhibitor (from Ambion RetroScript Kit: 1710, $206.00/kit)
• Reverse Transcriptase (M-MLV) (5x RT Buffer from Ambion RetroScript Kit: 1710, $206.00/kit)
• 10x PCR Reaction Buffer (from Ambion RetroScript Kit: 1710, $206.00/kit)
• 50 mM MgCl₂ (from Ambion RetroScript Kit: 1710, $206.00/kit)
• SuperTAQ DNA Polymerase (Ambion, 2050, $48.00/50 U or 2052, $190.00/250U)
• 18S rRNA PCR Primer Pair (Ambion kit, 1716 $155.00/kit)
• 18S PCR Competimers (Ambion kit, 1716 $155.00/kit)
• Sybr Green Stain (FMC Bioproduct, 50513, $146.00/box)

Protocol:

Using the cDNA from the previous RT PCR, optimal cycles were determined for gene of interest. The optimal 18S rRNA PCR Primer:Competimer ratio will need to be optimized per gene primer pair (start with 1:9, 2:8, 3:7 and reduce ratio again if rare primer)

1. Prepare of Primer Mixes (for desired gene) per reaction:

   Forward Primer 2 µl
   Reverse Primer 2 µl
   Total Primer Mix 4 µl

2. Prepare of 10s rRNA PCR Primer:Competimer Mix (e.g., 1:9 Ratio):

   18 S rRNA PCR Primer 1µl
   Competimer 9 µl
   Total 1:9 Mix 10 µl

3. Prepare PCR Reaction Cocktail (ideal to include 4 controls):

   per reaction:
   10x Complete PCR Buffer 5 µl
   2.5 mM dNTP 4 µl
   Taq Polymerase 0.2 µl
   18 S Primer:Competimer Mix 4 µl
   RNase-free H₂O 4 µl
   Total 45 µl
   Mix by pipetting up and down, centrifuge briefly

For your Controls:

Aliquot 45 µl into 4 PCR (0.2 ml) tubes marked C1, C2, C3, C4 (or aliquot 180 µl (45 µl x 4) into a tube with 16ul 18 S primer pair and aliquot 49 µl into 4 tubes)

Add 4 µl 18 S Primer pair to each tube then add 1 µl of the following:
C1 = 1 µl of (+) RT Control cDNA
C2 = 1 µl of (-) RT Mock Control cDNA
C3 = 1 µl of (-) RT LAU Control cDNA
C4 = 1 µl of (-) PCR Control (ddH₂O)
Total reaction mix/tube per tube = 50 µl

Add 4 µl of gene specific primer mix per cocktail mix. Mix well by pipetting up and down.

Aliquot 49 µl into a PCR tubes

Add 1 µl of appropriate cDNA. and mix by pipetting up and down, centrifuge briefly and keep on ice.

Close tube lid tightly and put into PCR machine at 95°C.

Run the tubes at the pre-determined optimal cycles per gene.

Perform PCR under the following conditions:

1. 95°C for 3 minutes
   start cycles:
2. 95°C for 45 seconds
3. 58°C for 45 seconds (check annealing temp for gene)
4. 72°C for 45 seconds
   REPEAT for OPTIMAL CYCLES (pre-determined)
5. 72°C for 10 minutes
6. 4°C Forever

Remove tubes at the appropriately designated cycle and kept on ice or at 4°C until gel can be run.

Analyze 10 µl PCR Product by mixing with 2.5 µl 5x dye in a 2% agarose gel with 3.5 µl Ethinium Bromide in 1x TAE buffer.

• Run for 1.5 hours (or until ready) at 100V or overnight at 25V.
• Stained gel with Sybr Green (250 µl 1x TAE and 25 µl Sybr Green) for 1.5 hours while shaking.

1. Observe gel under UV light and photograph the image. Also, scan the gel under STORM Fluorimager to be able to analyze quantitative differences.

• ddH₂O

• Phenol, water-saturated and buffered (pH 7.5)

• Chloroform

• G25 column

• Linear acrylamide

• 3M Sodium Acetate pH 7.5 and pH 5.2

• Ethanol

• T7 polymerase with 5x reaction buffer and DTT

• Nucleosides

• RQ1 RNase-free DNase

Protocol:

A. Prepare template

1. Adjust volume of template PCR or linearized plasmid to 200 µl with ddH₂O

2. Phenol/Chloroform extract 3x with 100 µl P/C pH7.5

3. Pass through G25 column

4. Add:

   1. 1 µl linear acrylamide

   2. 30 µl Sodium Acetate pH 7.5

   3. 600 µl 100% Ethanol

5. Mix, precipitate at -80°C for 15 minutes.

6. Spin 12 minutes at 14,000 rpm

7. Wash pellet with 70% Ethanol

8. Wash pellet with 100% Ethanol

9. Dry pellet for 10 minutes

10. Dissolve pellet in 11 µl ddH₂O

11. Use 1 µl template to determine concentration.

12. Use 1.5 µg template in 10 µl ddH₂O for transcription reaction.

B. In vitro transcription reaction

1. Make reaction mix:

   1. 38 µl ddH₂O

   2. 20 µl 5x Buffer

   3. 10 µl 100 mM DTT

   4. 5 µl 10 mM rGTP

   5. 5 µl 10 mM rATP

   6. 5 µl 10 mM rUTP

   7. 5 µl 10 mM rCTP

   8. 1 µl RNase Inhibitor

   9. 1 µl T7 polymerase

2. Vortex, spin briefly

3. Add 10 µl template DNA, mix, spin briefly

4. Incubate for 1 hour at 40°C

C. Destroy DNA, purify RNA

1. Add 1 µl RQ1 RNase-free DNase

2. Incubate for 15 minutes at 40°C

3. Add 106 µl ddH₂O

4. Sequentially add the following:

   1. 200 µl Phenol (water-saturated)

   2. 40 µl Chloroform

5. Vortex, spin for 5 minutes at 14,000 rpm

6. Pass the upper aqueous phase through G25 column

7. Precipitate with:

   1. 1 µl linear acrylamide

   2. 30 µl 3M Sodium Acetate pH5.2

   3. 600 µl 100% Ethanol

8. Mix, precipitate at -80°C for 15 minutes.

9. Spin 15 minutes at 14,000 rpm

10. Wash pellet with 70% Ethanol

11. Wash pellet with 100% Ethanol

12. Dry pellet for 10 minutes

13. Dissolve pellet in 11 µl ddH₂O

14. Use 1 µl to determine RNA concentration

Svetlana Mikheeva, 13 August 1999

**
**

MATERIALS

Equipment

- Single channel pipette: 0.5-10 µl, 10-100 µl, 200-1000 µl, Eppendorf

- Vacuum manifold, Millipore

- Speedvac

- Adjustable waterbaths (37° C, 42° C, 55° C, 100° C)

Disposables

- 96-well Multiscreen-FB filter plate, Millipore, MAFB NOB 10

- Catch plate, VWR, 622409-108

- Pipette tips: 1-10 µl, 2-20 µl, 20-200 µl, 200-1000 µl, ART

- Tubes: 1.5ml

Reagents

- Anchored dT25, dT₂₃VN, 8 µM stock GibcoBRL

- Random 9-mers, NNNNNNNNN, 1 µg/µl stock Gibco-BRL

- GFP poly A⁺ RNA, prepared from plasmid pSP64-GFP

- SuperScript II, GibcoBRL, 200 U/µl, 18064-022 (includes 5X First Strand Buffer and DTT)

- Nucleotides, Pharmacia 27-2035-02, 100mM each*

- 5-(3-Aminoallyl)-2'-deoxyurindine 5'-triphosphate sodium salt (AA-dUTP), Sigma A-0410

- RNase Inhibitor, Boehringer Mannheim 799 017, 40U/ml

- Buffers, Sigma, 2.5M NaOH, 2M MOPS, 1M Tris-HCl pH 7.5 8.5

- 4M Hydroxylamine, Sigma H-2391

- 0.1M NaBicarbonate pH 9.0

- Monofunctional NHS-ester Cy3/Cy5, APBiotech PA23001/PA25001; resuspend each tube in 72µl H₂O, aliquot 4.5µl into 16 tubes, dry in speedvac

- Millipore Binding Buffer (5.3M Gua-HCl in 150mM KAc, pH 4.8)

- Millipore Wash Solution (80% Ethanol)

- Slide pretreatment buffer, 5XSSC/0.2%SDS

- Hybridization Buffer (50% Formamide, 5X SSC, 5X Denhardt�s, 0.1%SDS, 100µg/ml ssDNA (Sigma), 100µg/ml COT-I DNA (BRL), 100µg/ml polyA₇₂)

- Wash buffers, 1XSSC/0.2%SDS, 0.1XSSC/0.2%SDS, 0.1XSSC

* Nucleotide mix (20X): 10mM GTP, 10mM ATP, 10mM CTP, 4mM TTP, 4mM AA-dUTP

PROTOCOL

Reverse Transcription

- Mix together:

2 µg poly A⁺ RNA (or 50mg total RNA)

2 µl oligo dT₂₃VN (8 mM)

2 µl random 9-mers (1mg/ml)

2.5 ng GFP poly A⁺ RNA

10.5 µl total volume

- Incubate at 70° C for 10 minutes, chill on ice 30 seconds, spin

- Add the following:

4 µl 5X First Strand Buffer

2 µl DTT (0.1M)

1 µl Nucleotide Mix (10mM G/A/C, 4mM T, 4mM AA-dUTP)

1 µl H₂O

0.5 µl RNase Inhibitor

- Mix contents of tube gently and incubate at RT for 10 minutes

- Add 1 µl SuperScriptII

20 µl total reaction volume

- Mix contents of tube gently and incubate at 42°C for 2 hours

RNA hydrolysis and purification of cDNA

- Add 2 µl 2.5 M NaOH

- Incubate at 37°C for 10 minutes

- Add 10 µl 2 M MOPS

- Add 200 µl binding buffer to probe and mix

- Dispense into glass fiber filter plate

- Place filter plate on top of vacu-system and apply vacuum.

- Wash 6x with 80% fresh Ethanol

- Place filter plate on top of catch plate along with centrifuge alignment frame

- Do one dry spin to remove residual ethanol (3500 rpm, 5�)

- Add 50 µl H₂O. Incubate 1 minute at RT

- Place filter plate on top of a clean catch plate along with a centrifuge alignment frame and spin (3000 rpm, 5�)

- Repeat with another 50 µl H₂O

- Scan probe at OD 260nm, 550nm and 650nm.

- Dry in Speedvac until volume is less than 5µl (~45 minutes @ 50�C)

Coupling to NHS-ester Cy dyes

- Adjust volume of sample to 4.5µl

- Resuspend mono-functional NHS-ester Cy3 or Cy5 dye aliquot in 4.5µl of 0.1M NaBicarbonate Buffer pH 9.0

- Mix dye and cDNA

- Incubate 1 hr at RT (Note: there is no advantage to incubating longer)

Quenching the Reaction and Removal of uncoupled Cy dyes

- Add 4.5µl of 4M Hydroxylamine

- Add 16.5µl of H₂O to bring the volume to 30µl

- Add 150µl of Millipore binding buffer, purify as above in Millipore 96 well plate, elute twice in 50µl aliquots of H₂O

- Scan probe at OD 260nm, 550nm and 650nm and calculate probe yield, incorporated Cy-dye and specific activity

- Purify probe over G50 column (prespin column (3000 rpm 1�), transfer to new tube, apply probe, spin (3000rpm 2�), collect flow-through).

- Dry purified probe down in speedvac (~1.5 hours at 50 °C)

Hybridization

- Slide pretreatment: submerge mirrored slides for 40 minutes in 5XSSC/0.2%SDS at 55 °C (waterbath), rinse slide by two quick dips in ddH₂O, air dry. Rinse coverslip in ddH₂O and ethanol, air dry

- Resuspend dried down probe in Hybridization Buffer (25µl/reaction)

- Boil probe for 3 minutes, ice 30 seconds, spin briefly

- Apply probe to slide and cover with coverslip

- Incubate overnight (14-16 hrs) at 42 °C in humid chamber

- Preheat wash buffers to 55 °C

- Remove coverslip by immersing slide in 1XSSC/0.2%SDS

- Wash in 1XSSC/0.2% SDS for 10 minutes at RT

- Wash twice in 0.1XSSC/0.2%SDS at RT for 10 minutes

- Wash twice in 0.1XSSC at RT for 1 minute

- Rinse by two quick dips in ddH₂O, dry with compressed air

- Scan (PMT 700, green (532nm), filter 1; PMT700, red (633nm), filter 2; width 10mm, length 60mm).

* Nucleotide mix: always use 10mM dGTP/dATP/dTTP and 1mM dCTP; unlabeled dCTP used in 1:1 ratio with Cy-labeled dCTP.

CY-DYE PROBE SYNTHESIS (17 February 2001)

Protocol:

1. Mix together:

2 µg mRNA

2 µl oligo dT₂₃VN (8 µM)

2 µl random 9-mers (1 µg/µl)

1 µl GFP mRNA (1 ng/µl)

10.5 µl total volume

1. Heat to 70°C for 10 minutes.

2. Chill on ice for 30 seconds.

3. Centrifuge briefly.

4. Add the following:

4 µl 5X First Strand Buffer

2 µl DTT (0.1 M)

1 µl Nucleotide Mix (10 mM G/A/T, 1 mM C)

1 µl Cy3- or Cy5-dCTP (1 mM)

0.5 µl RNase Inhibitor

Note: when doing multiple reactions, make up premix containing all but Cy-dyes and aliquot 7.5 µl premix to each tube before adding Cy-dyes.

1. Mix contents of tube gently and incubate at RT for 10 minutes.

2. Add

1 µl SuperScriptII

20 µl total reaction volume

1. Mix contents of tube gently and incubate at 42°C for 2-3 hours.

2. Denature cDNA/mRNA hybrid as follows:

Add 2 µl 2.5 M NaOH

Incubate at 37°C for 10 minutes

Add 10 µl 2 M MOPS

1. Add 200 µl binding buffer to probe and mix.

2. Dispense into glass fiber filter plate.

3. Place filter plate on top of vacu-system and apply vacuum.

4. Wash 6x with 200 ml 80% Ethanol.

5. Place filter plate on top of catch plate along with centrifuge alignment frame.

6. Do one dry spin to remove residual ethanol (3500 rpm, 5 min).

7. Add 50 µl 10 mM Tris-HCl, pH 8. Incubate 1 minute at RT.

8. Place filter plate on top of a clean catch plate along with a centrifuge alignment frame and spin (3000 rpm, 5 min).

9. Repeat with another 50 µl Tris-HCl.

10. Scan probe at OD 260 nm, 550 nm and 650 nm and calculate probe yield, incorporated Cy-dye and specific activity.

11. Purify probe over G50 column (prespin column (1000x g 1 min), transfer to new tube, apply probe, spin (1000x g 2 min), collect flow-through).

12. Dry purified probe down in speedvac (1 hour at 50°C).

13. Rinse slides and coverslips by two quick dips in ddH₂O, air dry.

14. Resuspend dried-down probe in 25 µl volume of hybridization solution (50% formamide, 5x SSC, 5x Denhardt�s, 0.1% SDS, 100 µg/ml poly A₇₂, 100 µg/ml human CotI DNA; pass through 0.2 µm filter).

15. Boil probe for 3 minutes, ice 30 seconds, spin briefly.

16. Combine with appropriate reaction; total volume is now 50 µl.

17. Apply probe to slide and cover with coverslip.

18. Incubate overnight (14-16 hrs) at 42°C in humid chamber.

19. Preheat wash buffers to 55°C.

20. Remove coverslip by immersing slide in 1x SSC/0.2% SDS.

21. Wash in 1x SSC/0.2% SDS for 10 minutes at RT.

22. Wash twice in 0.1x SSC/0.2% SDS at RT for 10 minutes.

23. Wash twice in 0.1x SSC at RT for 1 minute.

24. Rinse by two quick dips in ddH₂O

25. Dry with compressed air.

26. Scan (PMT 600, green (532nm), filter 1; PMT650, red (633nm), filter 2; width 10mm, length 60mm).

PS. Name Fluorescence Solution

Cy3 = green = pink

Cy5 = red = blue

20X SSC (3M NaCl, 0.3M NaCitrate, pH 7.0):

- adjust pH to 7.0 with 1M HCl

100X Denhardt:

- filter sterilize

- store at �20 °C in 25ml aliquots

Hyb Solution (5X SSC, 5X Denhardt, 50% Formamid, 1% SDS)

Protocol:

1. Rinse gel with ddH₂0, 3X.
2. Soak in 20X SSC for 45 minutes.
3. Take photograph of gel.
4. Soak nylon membrane -cut to size- in ddH₂O for 5 minutes.
5. Put sponge in container, fill with 20X SSC halfway up sponge.
6. Put 3 20X SSC-soaked GB002-sheets on top of sponge.
7. Place gel on top, remove bubbles.
8. Cover with nylon membrane, remove bubbles.
9. Successively add 1 GB002, 4 GB003 and 4cm GB004.
10. Cover with glass plate and 0.4kg weight.
11. Transfer overnight.
12. Take structure apart, mark wells on membrane.
13. Visualize and mark rRNA bands and RNA MW ladder on membrane.
14. Rinse membrane in 2X SSC.
15. Dry on GB003 and wrap in plastic foil
16. UV cross-link (�Autolink� on Stratalinker).
17. Take photograph of flattened gel to assess transfer efficiency.
18. Pre-hybridize membrane for 4-20 hours in 5-10 ml Formamid (Pre-) Hybridization solution (FPH) at 42 °C.
19. Boil 100µl probe for 10 minutes (use 1 x 10⁶ cpm/ml FPH).
20. Cool on ice, spin.
21. Add to hybridization bag.
22. Incubate 20 hours at 42 °C.
23. Rinse in 2X SSC/0.1% SDS at RT, 3X.
24. Wash in 0.2X SSC/0.1% SDS for 15 minutes at 42 °C, 2X.
25. Wash in 0.1X SSC/0.1% SDS for 15 minutes at 65 °C, 2X.
26. Rinse in 2X SSC.
27. Wrap in plastic foil.
28. Expose to phosphoscreen.

Notes:

• After overnight exposure 5pg RNA can be detected with a probe labeled to a specific activity of 10⁹ dpm/mg.

• Probes labeled to ≥5 x 10⁸ dpm/µg should detect transcripts that represent 0.01% of mRNA population with a blot of 10 µg total RNA or 0.0002% of mRNA population with a blot of 3 µg poly(A)⁺ RNA.

• For stripping poor boiling 0.05% SDS on membrane, incubate for 10 minutes, repeat up to 3X. Rinse with 2X SSC.

Combine reagents listed below:

Primer VNG26 (30 µM) 1.0 µl

Template (100ng) 3.0 µl

10x PCR Buffer 5.0 µl

MgCl₂ (25mM) 3.0 µl

10X dNTP�s^* 5.0 µl

³² P dCTP (~10 µCi/µl) 10.0 µl

Water 22.8 µl

Taq polymerase 0.2 µl

Final Volume 50.0 µl

*10X dNTP stock: 2 mM dGTP/dATP/dTTP and 12 µM dCTP. Final concentration in the PCR reaction is: 0.2 mM dGTP/dATP/dTTP, and 0.0012 mM dCTP. Recipe: combine 5 µl 10mM dGTP, 5 µl 10mM dATP, 5 µl 10mM dTTP and 10 µl 30 µM dCTP for 25 µl total volume.

PCR cycles:

5 min at 95°C 1X

45 sec at 95°C

45 sec at 50°C 40X

4 min at 72°C

10 min at 72°C 1X

hold at 4°C

Purify the hot PCR product over a G50 column.

Count 1 µl: PCR counts should range ~2-3 x 10⁶ cpm/µl

(PRE) HYBRIDIZING THE PCR PROBE

Prehybridization:

1. Place blot in hybridization tube: add 6-10ml prehyb/hyb solution.

2. Place tube in 42°C, preheated, hybridization oven. Turn rotator to the �8� setting. Always include a balance tube.

3. Incubate for 3-6 hours.

Hybridization:

1. Boil labeled probe for 2 minutes. Chill on ice for 2 minutes, and add to the hybridization solution. Mix well before pouring the solution into the tube containing the blot. Use 6ml of solution with at least 2 x 10⁶ cpm/µl of probe (best to use entire probe for 6ml hyb, i.e. 2 x 10⁷ cpm/µl).

2. Pour prehybridization solution out of the hybridization tube and add the hybridization solution containing the probe.

3. Incubate in the hybridization oven at 42°C, rotating, overnight.

4. Rinse blot 1X quickly at room temp in 2xSSC/0.05%SDS.

5. Wash 1X for 20 minutes at room temp in 2xSSC/0.05% SDS.

6. Wash 1X for 30 minutes at 50°C in 0.1xSSC/0.1% SDS.

7. Wrap in Saran while the blot is still damp, and expose with a phosphor image screen.

Purchasing Bacterial Clones for Northern Verification:

Purchase the clones by their IMAGE ID number. The vendor is Research Genetics (phone number 1-800-533-4363) The cost is $45 for sequence verified clones.

Search engine with more clone info: http://www.resgen.com/resources/apps/cdna/index.php3 (Type in the IMAGE ID).

Image website: http://image.llnl.gov/

• 25-50ng of target DNA in no more than 25µl TE buffer.

• Ready.To.Go DNA Labelling Kit (-dCTP), Pharmacia, 27-9251-01

• ProbeQuant G-50 Micro Columns, Pharmacia, 27-5335-01

• ddH₂O

• 5ml scintillation fluid in vial.

Protocol

Probe synthesis

1. Reconstitute the contents of the Reaction Mix tube by adding 20µl ddH₂O. DO NOT MIX. Let sit on ice for 5-60 minutes.

2. Denature 25-50ng DNA by heating for 2-3 minutes at 95-100 °C. Immediately place on ice for 2 minutes, then centrifuge briefly.

3. Add the following to the reconstituted Reaction Mix tube:

Denatured DNA (25-50ng) 25µl

[a-32P]dCTP (3000 Ci/mmol) 5µl (50µCi)

ddH₂O to total of 50µl

4. Mix by gently pipetting up and down several times. Bubbles may be removed by a pulse centrifugation.

5. Incubate at 37 °C for 5-15 minutes. (Difficult templates may require up to 30 minutes.)

Note: The Reaction Mix contains dATP, dGTP, dTTP, FPLC-pure Klenow fragment (4-8 units) and random oligonucleotides, primarily 9-mers.

Probe purification

6. Resuspend the resin in spin column by vortexing.

7. Loosen the cap one-fourth and snap off the bottom closure.

8. Place the column in a 1.5ml screw-cap tube.

9. Pre-spin the column for 1 minute at 735 x g (3500rpm).

10. Place the column in a new 1.5ml tube and slowly apply 50µl of the sample to the top-center of the resin without disturbing the resin-bed.

11. Spin the column at 735 x g for 2 minutes. The purified probe is collected in the bottom of the support tube. Cap the tube. Store at �20 °C until use.

Note: For a force of 735 x g the appropriate speed can be calculated from the following formula: rpm = (1000) (657/r)^½ . For example with a rotor radius of 73mm, the appropriate speed would be 3000rpm.

Probe quantification

12. Resuspend 1µl of purified probe in 50µl ddH₂O.

13. Transfer mixture to a vial with 5ml scintillation fluid.

14. Count the samples (as user 7, T-wing).

Note: this protocol should label 25-50ng of DNA to 1 x 10⁹ dpm/µg. Yields are usually 10⁶ cpm/µl.

Probe qualification

15. Run 20,000 cpm probe on a 1% Agarose gel.

16. Place gel on 4 pieces of Whatman paper.

17. Cover just the gel with piece of Saran Wrap.

18. Dry down gel at 65 °C for 1 hour.

19. Expose phosphoscreen.

Note: PCR products should give one sharp band.

Reduced Formaldehyde denaturing gel (1% Agarose, 1x MOPS, 1.9% di-F)

25x MOPS (2 M MOPS, 500 mM NaOAc, 10 mM EDTA)ddH₂O

Denaturing Buffer (1x MOPS, 50% Formamide, 2% Formaldehyde)

Blue Juice (50% Glycerol, 0.27% BPB/XC, 1.3 mM EDTA)

Ethidium Bromide (1mg/ml)

� Blue Juice and Ethidium Bromide are added in 2:1 ratio.

� 100 ml gel: 50 µl sample (6 µl RNA + 30 µl denaturing buffer + 9 µl BJ/EB)

Protocol:

1. Dissolve agarose in MOPS and H₂O, cool to handwarm.

2. Add 37% di-Formaldehyde, poor gel, let solidify.

3. Flush wells after removal of comb.

4. Running buffer is 1x MOPS.

5. Add denaturing buffer to RNA samples in 5:1 ratio.

6. Incubate at 65°C for 15 minutes.

7. Cool on ice.

8. Add Blue Juice/Ethidium Bromide (2:1) to samples in 1:4 ratio.

9. Load on gel (max. 15 µl on 20ml gel, max. 50 µl on 100ml gel).

10. Run for 3 hrs at 75V.

Notes:

- Run 6 µg of RNA MW ladder along with samples.

- Optional: run ³²P labelled l/HindIII (10000 cpm) with samples.

1. Put 400 µl RNA gel matrix (red dot ●) and 4 µl RNA dye concentrate (blue dot ●) in a 1.5ml tube.

2. Vortex well. Transfer to spin filter.

3. Spin 10 minutes at 3000rpm. Protect from light. Store at 4°C. Use within 3 weeks.

Preparing ladder and samples:

1. Denature 1.5 µl RNA ladder for 3 minutes at 70°C. Chill on ice. Spin.

2. Mark 0.6 ml tubes “ladder” and “1” to “12”

3. Pipette 5 µl sample buffer (white dot ○) in each tube.

4. Pipette 1 µl denatured RNA ladder in tube marked “ladder”.

5. Pipette 1 µl of each sample into the appropriately marked tubes.

6. Vortex each tube for 5 seconds.

7. Spin tubes for 10 seconds.

8. Store on ice until ready to load samples.

Loading Gel-Dye Mix:

1. Put chip on preparation stand.

2. Pipette 9 µl gel-dye mix in circled G well

3. Pull back syringe-plunger to 1ml mark.

4. Put syringe tip around well to form tight seal.

5. Press plunger to 0.2ml and hold for 30 seconds.

6. Pull back plunger to the 1ml mark while maintaining the seal.

7. Remove syringe from well.

8. Pipette 9 µl gel-dye mix in other 2 G wells.

Loading the ladder and samples:

1. Pipette the 6 µl of ladder-tube in ladder-well

2. Pipette the 6 µl of each sample-tube in the 12 µl sample wells.

3. Treat bioanalyzer-electrodes for 30 seconds (no longer) with ZAP.

4. Treat bioanalyzer-electrodes for 30 seconds (no longer) with ddH₂O.

5. Dry bioanalyzer-electrodes for 10 seconds (no longer).

6. Insert the loaded chip into the bioanalyzer within 5 minutes after loading.

Reagents:

Agilent 2100 Bioanalyzer Reagent and Chip Kits

For quantitation of RNA samples, an RNA 6000 ladder must be purchased from Ambion, #7152.

Short Protocol:

1. Dissolve 5mg Acrylamide in 200 µl TE

2. Add 1 µl 10%APS

3. Add 1 µl TEMED

4. Allow to polymerize (incubate as long as you like, can heat to 37°C to increase rate): forms a jelly mass.

5. Add 2.5 volumes ethanol.

6. Spin 5-6 minutes.

7. Speed vac to dry.

8. Resuspend 500 µl ddH₂0 (i.e. 10 µg/µl). Takes a while to resuspend (may leave at RT overnight). Subsequent pellets are easy to resuspend.

9. Use 10-20 µg for a precipitation.

Linear poly-acrylamide can be used as an efficient neutral carrier for precipitating nucleic acids with ethanol (Gaillard, C and Strauss, F. Nucleic Acids Res. 18, 378). Alhough glycogen is often used as carrier in ethanol precipitation, it has been shown that glycogen inhibits the activity of an ssDNA-binding protein on which linear polyacrylamide has no effect. Disadvantage using linear polyacrylamide as carrier is that the pellet of polyacrylamide does not stick tightly on the bottom of microfuge tube. Be careful not to discard pellet when you remove supernatant.

Extended Protocol:

1. Prepare a 5% acrylamide solution without bis-acrylamide in 40 mM Tris-HCl (pH. 8), 20 mM sodium acetate, 1 mM EDTA.

2. Add ammonium persulfate to a final concentration of 0.1% (w/v).

3. Add 1/1000 volume of TEMED, and let polymerize for 30 min at room temperature.

4. When the solution has become viscous, precipitate the polymer with 2.5 volumes of ethanol.

5. Recover the polymer by centrifugation, and dissolve the pellet in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA to make a final polyacrylamide concentration of 5 mg/ml.

6. The linear polyacrylamide solution can be stored in the refrigerator for several years.

7. Add 10-20 micrograms of the linear polyacrylamide into nucleic acid solution before ethanol precipitation.

8. Picogram amounts of nucleic acids longer than 20 base pairs can be precipitated without loss.

• Oligo (dT)-cellulose Type 7 (Pharmacia)

• Polypropylene chromatography columns (BioRad)

• 0.1 N NaOH/5 mM EDTA

• 2x Column Loading Buffer (40 mM Tris pH 7.5, 1 M LiCl, 2 mM EDTA, 0.2% SDS)

• dH₂0

• 4 M LiCl

• Linear acrylamide (5 mg/ml)

• 100% EtOH

Protocol:

1. Pour a small amount of Oligo (dT)-cellulose powder into a conical tube and suspend in 1X Column Loading Buffer.

2. Transfer into the column that is sitting in a 15ml conical tube (up to 10mg RNA can be selected per ml of oligo (dT). Oligo (dT)-cellulose packed to 0.4ml for 1mg total RNA gave very good yields. Expect ~15-50 µg poly A+ RNA per mg of total RNA.

3. Wash with 10 volumes each

   1. sterile dH₂O

   2. 0.1N NaOH/5mM EDTA

   3. sterile dH₂O

4. Wash with 5 volumes 0.1x Column Loading Buffer.

5. Wash with 10 volumes dH₂O.

6. With pH paper, check to make sure the pH of the column effluent is less than 8.

7. Wash the column with 1x Column Loading Buffer, then replace the catch tube with a new one.

8. In the meantime, prepare the RNA sample:

   1. dissolve in 500 ml dH₂O

   2. heat at 65^oC for 5 minutes

   3. add an equal volume of 65^oC 2x Column Loading Buffer

   4. let cool to RT on benchtop

9. Apply RNA sample to column.

10. Collect the flow through, heat again to 65^oC for 5 minutes, allow to cool to almost RT and reapply to column.

11. Collect the effluent; this is the poly A- fraction.

12. Replace the catch tube and wash with 10 volumes of 1x Column Loading Buffer.

13. Replace the catch tube again and elute the poly A+ RNA with 2 ml 65^oC dH₂O.

14. Spin at 2000 rpm, 4^oC for 10 minutes to remove contaminating gel.

15. Read O.D. 260/280 and calculate concentration.

16. Ethanol precipitate with

    1. LiCl added to 0.2M (1:4)

    2. 100 µg linear acrylamide (carrier)

    3. 2.5 volumes 95% EtOH.

    Always do the precipitation overnight.

17. Regenerate the column by sequential washing with 10 volumes each of 0.1 N NaOH/5 mM EDTA, dH₂O and 1x Column Loading Buffer. Columns may be stored at -20 °C and used up to 3 times, depending on the amount of RNA selected.

Note: If you must break the 2 ml into aliquots for eppendorf tubes, use 20 µg linear acrylamide per aliquot.

For mRNA isolation

- Binding Buffer OBB

• 20 mM Tris-Cl pH 7.5, 1 M NaCl, 2 mM EDTA, 0.2% SDS

- Oligotex Suspension

• 10% (w/v) in 10 mM Tris-Cl pH 7.5, 500 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% NaN₃ (Qiagen cat.no. 79000)

- Wash Buffer OW2

• 10 mM Tris-Cl pH 7.5, 150 mM NaCl, 1 mM EDTA

- Elution Buffer OEB

• 5 mM Tris-Cl pH 7.5

- Small spin columns

• RNase-free spin columns (Qiagen cat.no. 79523)

For mRNA precipitation

- 4M LiCl

- Linear acrylamide (5 mg/ml)

- 100% EtOH

Oligotex mRNA kits Mini (70022, 250 µg), Midi (70042, 250 µg-1mg) and Maxi (70061, 1-3mg)

B. Protocol:

1. Dilute no more than 250 µg total RNA in 250 µl ddH₂O.

Add 250 µl OBB (pre-heated at 70°C).

Add 15 µl Oligotex (pre-heated at 37°C).

Mix by pipetting up and down.

Incubate at 70°C for 3 minutes.

Incubate at RT for 10 minutes.

Spin at 14,000 x g for 2 minutes.

Remove supernatant and save for second extraction (step 20).

Resuspend pellet in 400 µl OW2 by pipetting up and down.

Transfer to spin column in clean 1.5ml tube.

Spin at 14,000 x g for 1 minute. Discard flow-through.

Transfer spin column to clean 1.5ml tube.

Add 400 µl OW2.

Spin at 14,000x g for 1 minute. Discard flow-through.

Transfer to spin column in clean 1.5 ml tube.

Add 100 µl OEB (pre-heated at 70°C).

Resuspend Oligotex by pipetting up and down.

Spin 14,000x g for 1 minute.

Save eluted mRNA on ice.

Optional: resuspend Oligotex with supernatant from step 7 and transfer to 1.5 ml tube. Repeat extraction from step 4. Combine eluted mRNA.

Read OD_260/280 and calculate concentration.

Run 50-200 ng mRNA on Bioanalyzer.

Ethanol precipitate with

1. LiCl added to 0.2 M (1:20)

2. 20 µg linear acrylamide (carrier)

3. 2.5 volumes 100% EtOH.

Store at -70°C.

• Guanidine Thiocyanate (Fluka)

• 1 M Sodium Citrate (pH 7.0)

• 10% Sarcosyl

• 14 M 2-β Mercaptoethanol

• Rnase free H₂O

• 2 M Sodium Acetate (pH 5.2)

• water saturated Phenol

• 49:1 Chloroform/Isoamylalcohol

• Isopropanol

• 75% Ethanol

• DNase I (Promega)

• 10x reaction buffer (400 mM Tris-HCl, pH 7.9; 60 mM MgCl₂; 100 mM NaCl; 100 mM CaCl₂; 1 mM DTT)

Solutions

* β-Mercaptoethanol should be added to the amount of solution D that is to be used that day. Make up solution D without β-Mercaptoethanol and then add 7.2 µl/ml of solution D as needed. Solution D without β-Mercaptoethanol can be stored at room temperature for 1 month.

Protocol:

1. Lyse cells in solution D (100 µl/1 X 10⁶ cells).

2. Sequentially add the following:

   0.1 Vol 2M Sodium Acetate, mix

   1 Vol Phenol

   0.2 Vol Chloroform/Isoamylalcohol

3. Vortex 10 seconds.

4. Incubate on ice 15 minutes.

5. Centrifuge 30 minutes at 10K, 4°C.

6. Transfer aqueous (top) phase to new tube.

7. Precipitate with 1 volume Isopropanol at least 1 hour, -20°C.

8. Centrifuge 30 minutes at 10K, 4°C

9. Dissolve pellet in 300µl solution D.

10. Transfer to Eppendorf tube.

11. Precipitate with 1 volume Isopropanol at least 30 minutes, -20°C.

12. Centrifuge 30 minutes at 14K in Eppendorf tabletop centrifuge.

13. Wash pellet with 75% Ethanol.

14. Centrifuge 10 minutes at 14K.

15. Dry and resuspend in appropriate RNAse free reagent (ddH₂O, poly A selection buffer).

16. Read O.D. 260/280.

17. Set up DNase I reaction in 1x buffer (10 U/250 µg RNA in 200 µl).

18. Incubate at 37°C for 1 hour.

19. Sequentially add the following:

    0.1 Vol 2 M Sodium Acetate, mix

    1 Vol Phenol

    0.2 Vol Chloroform/Isoamylalcohol

20. Vortex and centrifuge 15 minutes at 14K.

21. Remove aqueous (top) phase to new tube.

22. Precipitate with 1 volume Isopropanol at least 30 minutes, -20°C.

23. Centrifuge 30 minutes at 14K.

24. Wash pellet with 75% Ethanol.

25. Centrifuge 10 minutes at 14K.

26. Dry and resuspend in appropriate RNase free agent.

27. Read O.D. 260/280 and calculate % yield.

28. Aliquot, store at �70°C under alcohol.

Chomczynske, P. and Sacchi, N., Analytical Biochem. 162: 156-159, 1987.

• Guanidine Thiocyanate (Fluka, FW=118.2, 250 g, 50990, $77.15)

• Citric Acid (Sigma, FW=294.1, 500 g, C-8532, $14.25)

• Sarkosyl (Sigma, FW=293.4, 50 g, L-9150, $14.75)

• 2-β Mercaptoethanol (Sigma, FW=78.13, 100 ml, M-7522, $15.40)

• RNase free ddH₂O (Sigma, 1 L, W-4502, $23.55)

• 3M Sodium Acetate Buffer (pH 5.2) (Sigma, 100 ml, S-7899, $11.90)

• Phenol (Boehringer Mannheim, 500 g, 1814303, $88.00)

• Chloroform (UW Stores, 4 L, 0011-555, $36.14)

• Isoamyl Alcohol (Sigma, FW=88.15, 500 ml, I-9392, $22.65)

• Isopropanol (UW Stores, 4 L, 0014-620, $22.08)

• 75% and 100% Ethanol

• 1 M Tris-HCl (pH 7.6) (Sigma, 1L, T-2788, $0.00)

• 20cc Syringe (UW Stores, 40/box, 0056-590, $11.12)

• 18Gx1-½ Needle (UW Stores, 100/pack, 0053-560, $4.05)

* β Mercaptoethanol should be added to the amount of solution D that is to be used that day. Make up solution D without β Mercaptoethanol and then add 7.2 µl/ml of solution D as needed. Solution D without β Mercaptoethanol can be stored at room temperature for 1 month.

Chomczynske, P. and Sacchi, N., Analytical Biochem. 162: 156-159, 1987.

Protocol:

1. Lyse cells in solution D (200 µl/l X 10⁶ cells).

2. Homogenize by passing lysate five times through 18-gauge needle with syringe. Store at �70 °C or continue.

3. Sequentially add the following:

0.1 volume 3 M Sodium Acetate (pH 5.2), mix

1.0 volume Phenol, water-saturated, mix

0.2 volume Chloroform/Isoamyl Alcohol (24:1) (fresh)

4. Vortex 10 seconds.

5. Incubate on ice 15 minutes.

6. Centrifuge 30 minutes at 10,000x g (brake slowly), 4°C.

7. Transfer upper aqueous phase to new tube.

8. Precipitate with an equal volume of Isopropanol for 30-60 minutes, -20°C.

9. Centrifuge 30 minutes at 10,000x g (brake max), 4°C.

10. Dissolve pellet in 600 µl solution D.

11. Transfer to Eppendorf tube.

12. Sequentially add the following:

60 µl 3 M Sodium Acetate (pH 5.2), mix

600 µl Phenol, water-saturated, mix

120 µl Chloroform/Isoamyl Alcohol (24:1) (fresh)

13. Vortex 10 seconds.

14. Incubate on ice 15 minutes.

15. Centrifuge 15 minutes at 14,000 rpm.

16. Transfer upper aqueous phase to new tube.

17. Precipitate with an equal volume of Isopropanol for 30-60 minutes, -20°C.

18. Centrifuge 5 minutes at 14,000 rpm.

19. Wash pellet with 200 µl 75% Ethanol.

20. Centrifuge 1 minute at 14,000 rpm.

21. Air dry and resuspend in 500 µl RNase-free ddH₂O.

22. Make 1:100 dilutions in 10mM Tris pH 7.6 to read OD_260/280.

23. Run 100-500 ng total RNA on Bioanalyzer.

24. Make 250 µg aliquots.

25. Store at �70 °C with 0.1 volumes 3M Sodium Acetate (pH 5.2) and 2.5 volumes 100% Ethanol.

Notes:

- Phenol is a poison and causes burns. Use gloves and eye protection.

- Carry out all steps at room temperature unless otherwise stated.

- The procedure can be carried out in sterile, disposable, 50 ml conical tubes (Falcon, Greiner).

- Do not let the RNA pellet dry completely as this greatly decreases its solubility.

- Dissolve the pellet in DEPC-treated water. Incubate 10 to 15 minutes at 55-60°C. Expected yield: 50 to 80 µg for 10 x 10⁶ cells.

- Water used for spectrophotometric measurement of RNA should have pH 7.5. Typically, distilled water has pH 6. Adjust water to a slightly alkaline pH by adding concentrated Na₂HPO₄ solution to a final concentration of 1 mM.

Solution D:

To Guanidine Thiocyanate bottle (250 g) add 293 ml ddH₂O preheated to 60°C, and 13.25 ml 1M Citric Acid (pH 7.0). Keep at 60°C and mix until dissolved. Add 26.5 ml of 10% (w/v) N-lauroylsarcosine (Sarkosyl) and water to 530 ml total volume. For use add 324 µl 2-β mercaptoethanol (2-ME) to 45ml solution.

Sodium acetate, 2M:

Add 16.42 g sodium acetate (anhydrous) to 40 ml water and 35 ml glacial acetic acid. Adjust solution to pH 4.0 with glacial acetic acid and dilute to 100 ml final with water (solution is 2 M with respect to sodium ions). Store up to 1 year at room temperature.

Water-saturated phenol:

Dissolve 50 g phenol in 50ml ddH₂0 at 60 to 65°C. Aliquot in 50 ml conical tubes wrapped in aluminium foil and store at �20°C. Do NOT use buffered phenol in place of water-saturated phenol.

Provided by Midi Kit (Qiagen, #75142, 10 samples, $90.00):

• 45 ml Buffer RLT (contains GITC, add 10ml β-Mercaptoethanol per ml RLT before use)

• 45 ml Buffer RW1 (contains GITC and ethanol)

• 11 ml Buffer RPE (concentrate, add 44 ml 100% ethanol before use)

• 10 ml RNase-free water

• 10 RNeasy Midi columns (RNA binding capacity up to 1mg each)

To be supplied by user:

• 20 cc Syringe (UW Stores, 40/box, 0056-590, $11.12)

• 14.3 M β-Mercaptoethanol (Sigma, FW=78.13, 100 ml, M-7522, $15.40)

• Ethanol (70% and 100%)

Protocol:

1. Wash cells once with PBS.

2. Lyse cells in buffer RLT (2 ml for up to 50 x 10⁶ cells).

3. Homogenize cell lysate immediately by passing 10 times through 18 gauge needle with syringe.

4. Add 1 volume (2 ml) of 70% Ethanol, mix thoroughly.

5. Transfer sample to RNeasy Midi column in 15 ml tube

6. Centrifuge at 3000-5000 x g for 5 minutes; discard flow-through.

7. Add 4ml of buffer RW1.

8. entrifuge at 3000-5000 x g for 5 minutes; discard flow-through.

9. Add 2.5ml of buffer RPE.

10. Centrifuge at 3000-5000 x g for 2 minutes; discard flow-through.

11. Add 2.5ml of buffer RPE.

12. Centrifuge at 3000-5000 x g for 5 minutes; discard flow-through.

13. Transfer RNeasy Midi column to clean 15 ml tube.

14. Add max. 250 µl RNase-free H₂O, incubate for 1 minute.

15. Centrifuge at 3000-5000 x g for 3 minutes

16. Add max. 250 µl RNase-free H₂O, incubate for 1 minute.

17. Centrifuge at 3000-5000 x g for 3 minutes; discard column.

18. Read O.D. 260/280.

19. Give sample to CEA for Bioanalyzer run.

Reagents:

* Nucleotide mix: use 10 mM dGTP/dATP/dTTP/dCTP.

** **

Protocol:

1. Mix together:

   200 ng poly A⁺ RNA (or 4-10 µg total RNA)

   1 µl oligo-dT/T7 (1 µg/µl)

   10 µl total volume

2. Heat to 70°C for 10 minutes.

3. Chill on ice for 30 seconds.

4. Centrifuge briefly.

5. Add the following (on ice):

   4 µl 5x First Strand Buffer

   2 µl DTT (0.1 M)

   1 µl dNTP mix (10 mM)

   1 µl Linear Acrylamide (0.1 µg/µl)

   1 µl RNase Inhibitor (40 U/µl)

   Note: when doing multiple reactions, make up premix containing all and aliquot 9 µl premix to each tube.

6. Mix contents of tube gently. Heat to 42°C for 1 minute.

7. Add:

   1 µl SuperScript II (200 U/µl)

   20 µl total volume

8. Mix contents of tube gently and incubate at 42°C for 1-2 hours.

9. Place on ice and keep cool while adding second strand components.

10. Add the following:

    30 µl 5x Second Strand Buffer (recipe below)

    3 µl dNTP mix (10 mM)

    4 µl E. coli DNA polymerase I (10 U/µl)

    1 µl E. coli RNase H (2 U/µl)

    1 µl E. coli DNA ligase (10 U/µl)

    91 µl ddH₂O

    150 µl total volume

11. Mix contents of tube gently and incubate at 16°C for 2 hours.

12. Add 3 µl 2.5M NaOH and incubate at 37°C for 10 minutes.

13. Add 150 µl buffered Phenol:Chloroform:Isoamylalcohol (25:24:1).

14. Mix by pipetting, spin 15K for 5 minutes.

15. Transfer aqueous phase to new tube.

16. Precipitate with 150 µl 5 M Ammonium Acetate and 1 ml 100% Ethanol.

17. Keep at -20°C for at least 15 minutes.

18. Vortex, spin 15K for 20 minutes at RT.

19. Add 100 µl 100% Ethanol, spin 15K for 5 minutes.

20. Resuspend dry pellet in 50 µl ddH₂O or 10mM Tris pH 7.5.

21. Pass through BioRad MicroBioSpin column (P-6 5bp, P-30 20bp). Mix column, remove cap, snap off bottom, spin 2 minutes at 1000x g, change collection tube, add sample, spin 4 minutes at 1000x g.

22. Dry purified probe down in speedvac to 16 µl or less.

23. T7 amplification kit: use all 16 µl in 40 µl reaction (double volume). Add sequentially (at RT):

    16 µl template DNA

    4 µl 10x T7 Reaction Buffer

    12 µl NTP mix (100 mM)

    4 µl DTT (100 mM)

    4 µl Ampliscribe T7 enzyme solution

    40 µl total volume

24. Incubate 42°C for 2-3 hours.

25. Add 110 µl H₂O and 150 µl water-saturated Phenol:Chloroform:Isoamylalcohol (25:24:1).

26. Mix by pipetting, spin 15K for 5 minutes.

27. Transfer aqueous phase to new tube.

28. Precipitate with 150 µl 5 M Ammonium Acetate and 1 ml 100% Ethanol.

29. Keep at -20°C for at least 15 minutes.

30. Vortex, spin 15K for 20 minutes at RT.

31. Add 100 µl 100% Ethanol, spin 15K for 5 minutes at 4°C.

32. Resuspend dry pellet in 30-50 µl ddH₂O.

33. Pass through BioRad MicroBioSpin column as in step 21.

34. Quantitate approximate aRNA yield by Bioanalyzer and spectrophotometer (1 µl).

Ready for CyDye probe synthesis. Label at least 5 µg of amplified RNA per reaction with 3-8 µg random hexamers. Yield ~20 µg aRNA from 200ng mRNA.

5x Second Strand Buffer Recipe:

Final Concentration Stock 10 ml

100 mM Tris pH 7.0 1.0 M 1.000 ml

450 mM KCl 2.0 M 2.250 ml

23 mM MgCl₂ 1.0 M 0.230 ml

50 mM (NH₄)₂SO₄ 1.0 M 0.500 ml

750 µM β-NAD 0.1 M 0.075 ml

ddH₂O - 5.945ml

• Making 0.1 M β-NAD: 0.25(g)/663.4(mw)/0.1(M)=3.768(ml). Add 3.768 ml ddH₂O to 250 mg β-NAD.

• Making 50 ml 1M (NH₄)₂SO₄: 132.14(mw) x 0.05(ml) x 1(M)=6.607 (g). Add 50 ml ddH₂O to 6.607 g (NH₄)₂SO₄.

• All the other buffers are from the Ambion Buffer Kit, 9010