18 June 2002

 

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.
  2. Mix together for each reaction:

    10x Complete PCR Buffer* 5.0 µl
    2.5 mM dNTP 4.0 µl
    Forward Primer of desired gene 2.0 µl
    Reverse Primer of same desired gene 2.0 µl
    Taq Polymerase 0.2 µl
    ddH2O 35.8 µl (or less to total volume*)
    total volume/sample 49.0 µl

    18 S Primer is the (+)RT control; for all other controls use the same primer as for the sample.

    For master mix, add 10% extra.

    * If you don't have Complete PCR Buffer, use 10x PCR Buffer, 1.5 µl MgCl2 and only 34.3 µl dd H2O; if you would like to vary the MgCl2 concentration, use 10x PCR Buffer and varying amounts of MgCl2 adjusting the total volume to 49 µl using dd H2O.

  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. Tube number: 3 4 5 6 7 8 9 10 11 12
    Cycle number: 17 19 21 23 25 27 29 31 33 35

  4. 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

Problem Solution

No Band

  • Decrease stringency= lower annealing temperature or increase MgCl2 concentration
  • Increase cycle number

     

Too many bands

  • Increase stringency: Higher annealing temp or decrease MgCl2
  • Decrease cycle number
  • Perform hot start PCR
  • Decrease primer and/or template concentration
  •  

Wrong size band

  • Raise annealing temp
  • Perform hot start PC
  •  

Primer-dimers

  • Set up reactions on ice and perform hot start PCR
  • Lower primer concentration (try 50-100 nM)
  •  

Band in negative RT lane

  • Treat with DNase free
  • Design primers over several exons
Department of Microbiology
School of Medicine
University of Washington
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