Introduction

Quantitation of first-strand cDNA synthesis is typically accomplished by incorporating a trace amount of a radiolabeled deoxynucleotide during the reverse transcription reaction. The protocol presented here provides an alternative, nonradioactive method for cDNA quantitation using OliGreen® ssDNA quantitation reagent (Molecular Probes) and a standard curve of random decamers. OliGreen is a fluorogenic dye that exhibits fluorescence when bound to single-stranded DNA. Because the dye also fluoresces when bound to RNA, the following procedure includes a step to degrade the RNA prior to cDNA quantitation.

For convenience, random decamers are used in this procedure to prepare the standard curve. The fluorescence enhancement of OliGreen bound to decamers is less than that of OliGreen bound to single-stranded cDNA. Ambion scientists have determined that this difference is 4-fold*, thus cDNA exhibits a 4-fold higher fluorescence than random decamers of the same concentration. This 4-fold difference is taken into account in the quantitation section of the protocol.

*Known concentrations of random decamers were compared to cDNA quantified by incorporation of a trace label.

Required Materials

• EDTA (Ambion Cat #9260G)
• RNase Cocktail (Ambion Cat #2286)
• Buffered Phenol:Chloroform:Isoamyl Alcohol (25:24:1) (Ambion Cat #9732)
• Chloroform
• Gel filtration spin columns designed to remove free nucleotides (e.g., Ambion's NucAway Columns, Cat #10070, or MicroSpin™ S-400 HR Column from Amersham Biosciences)
• 5 M NH₄OAc (Ambion Cat #9070G)
• 100% Ethanol
• TE buffer (Ambion Cat #9861)
• OliGreen Reagent
• Random Decamer Primers (Ambion Cat #5722G) for use as a standard

Protocol

A. RNA Degradation (to be performed after first-strand cDNA synthesis)

1. To your reverse transcription reaction, add EDTA to a final concentration of 6 mM. Heat at 90°C for 5 min. Chill on ice.
   
   
2. Add 1 µl RNase Cocktail and incubate at 37°C for 15 min.
   
   
3. Extract with an equal volume of buffered Phenol:Chloroform:IAA (not acid phenol). Transfer the aqueous phase (top layer) to a new 0.5 ml tube.
   
   
4. Add an equal volume of Chloroform. Vortex and spin as before. Transfer aqueous phase to a new tube.
   
   
5. Bring the volume to 100 µl with addition of TE buffer.

B. cDNA Purification

1. Prepare the spin column as per the manufacturer's instructions.

   For for a NucAway column:
   

   • Tap the column to settle the dry gel in the bottom of the spin column.
   • Hydrate the column with 650 µl of RNase-free water or the buffer of your choice
     
   • Cap, vortex, tap out air bubbles, and hydrate at room temperature 5 min to 15 min
     
   • (optional) Once rehydrated, these can be stored in the refrigerator up to 3 days.
     
   • Spin the column at 750 x g for 2 min. to remove excess interstitial fluid, keeping track of the orientation of the column in the rotor.
     
     

   For a MicroSpin S-400 HR column:

   • Vortex the spin column to resuspend the resin.
   • Remove the top cap and snap off the bottom of the column. Place the column in a collection tube.
   • Centrifuge the column for 1 min at 735 x g.
     
     
2. Transfer the column to a new collection tube. Carefully apply the RT reaction to the center of the tube.
   
   
3. Spin the column as per the manufacturer's instructions (750 x g for 2 min for a NucAway column; 735 x g for 2 min for a MicroSpin S-400 HR column). The sample will be collected in the collection tube.
   
   
4. Add 10 µl 5 M NH₄OAc and 110 µl buffered Phenol:Chloroform:IAA. Vortex thoroughly and spin 5 minutes in a microfuge at room temperature. Transfer the aqueous phase (top layer) to a new 0.5 ml tube.
   
   
5. Add an equal volume of Chloroform, vortex and spin as before. Transfer the aqueous phase to a clean 1.5 ml tube.
   
   
6. Add 275 µl 100% ethanol, and incubate at -20°C for 15 min.
   
   
7. Spin at maximum speed in a microfuge at 4°C for 20 min. Remove EtOH and let the pellet air dry for 3-5 min at room temperature.
   
   
8. Resuspend the pellet in 100 µl TE buffer and transfer to non-stick microfuge tube.
   
   
9. Quantify against standards as described below.

C. cDNA Quantitation by OliGreen

1. Thaw the OliGreen reagent.
   
   
2. Mix 4 µl OliGreen reagent with 1600 µl TE for the standards, plus an additional 1 µl OliGreen and 400 µl TE for each cDNA preparation to be assayed.
   
   
3. Dilute the Random Decamer Primers in TE buffer to 100 ng/µl. The Random Decamer Primers from Ambion are supplied at a concentration of 50 µM (156 ng/µl).
   
   
4. Prepare serial dilutions of the Random Decamer Primers. Recommended concentrations for the standards are 100 ng/µl, 33 ng/µl, 10 ng/µl, 3 ng/µl and 1 ng/µl.
   
   
5. Prepare one 3-fold dilution of each cDNA sample in TE buffer.
   
   
6. Pipet 1 µl of each standard into separate wells of a 96 well microplate. Place 1 µl of TE into another well as a no-DNA control.
   
   
7. Pipet 1 µl of the undiluted cDNA and 1 µl of the diluted cDNA into separate wells.
   
   
8. Add 200 µl of the diluted OliGreen solution to each standard and sample well. Mix by pipetting up and down.
   
   
9. Incubate for 5 min in subdued light.
   
   
10. Measure the fluorescence in a microplate fluorometer using an excitation wavelength of ~480 nm and an emission wavelength of ~520 nm.
    
    
11. Subtract the fluorescence value of the no-DNA control from that of all samples and standards.
    
    
12. Determine the concentration of the cDNA by comparing its fluorescence to that of the Random Decamer Standard curve and then dividing the cDNA concentration by 4.
    

OliGreen is a registered trademark of Molecular Probes, Inc.

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