18 June 2002
- Forward Gene Specific Primer (IDT)
- Reverse Gene Specific Primer (IDT)
- Random decamer primer (from Ambion RetroScript Kit: 1710, $206.00/kit or Ambion, 5722G, $50.00/80Âµl)
- 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)
Adjust concentration of RNA to 100 ng/Âµl (The RNA should be DNase treated or equivalent).
Set up two 500 Âµl 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.
For each tube mix together:
RNA (100-450ng) 2 Âµl
Ramdom decamer (in excess) 2 Âµl
H2O to equal total volume 8 Âµl
Total volume 12 Âµl
For a positive control use 2 Âµl of the RNA Control Template (Ambion).
Heat at 75Â°C for ten minutes; briefly centrifuge and keep on ice 30 seconds or until ready.
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 Âµl 5x RT Buffer 2 Âµl 2.5mM dNTP 4 Âµl 2.5mM dNTP 4 Âµl RNase Inhibitor (40 U/Âµl) 1 Âµl RNase Inhibitor (40 U/Âµl) 1 Âµl M-MLV Reverse Transcriptase 1 Âµl H2O 1 Âµl Total Volume 8 Âµl Total Volume 8 Âµl
Note: When doing multiple reactions, make up a master mix for all samples allowing 10% extra to permit for pipetting errors. Aliquot 8 Âµl/sample or control and add to the reaction mixture from step 2.
Add 8 Âµl of the RT Cocktail mix to the 12 Âµl of RNA mix. Mix by pipetting up and down, centrifuge briefly.
Incubate at 42Â°C for 1 hour.
Centrifuge briefly and store at -20Â°C until ready to continue.
Polymerase Chain Reaction
- For linear range PCR, we find it best to use 10 cycle-increments
- -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.
- For each of the controls add 1 Âµl of the following. | | | | | — | ————————— | ——————————————– | | 1a. | Positive RT Control-Mock | 1Âµl cDNA from RNA Control Template (Ambion) | | 1b. | Negative PCR Control | 1Âµl H2O | | 2a. | Negative RT Control-Mock | 1Âµl (-)RT Mock cDNA | | 2b. | Negative RT Control-Treated | 1Âµl (-)RT Treated cDNA |
For Linear Range
- For each sample, add 1 Âµl cDNA for a total of 50 Âµl.
- Distribute 50 Âµl each into 10 separate tubes with each representing a different cycle.
- Set and run the following PCR cycles:
- 95Â°C for 3 minutes
- 95Â°C for 45 seconds
- 58Â°C for 45 seconds
- 72Â°C for 45 seconds
REPEAT from step “b” 35 times
Note: Preheat thermocycler before placing the sample tubes in the machine.
- 95Â°C for 3 minutes
- Remove the appropriate tube after the designated cycle is over. Ideally you should run a final extension cycle in another thermocycler (10 minutes at 72Â°C). Put on ice for 1 minute. Keep at 4Â°C until ready to load into gel. Store PCR product at -20Â°C.
- Add 2.5 Âµl of dye to 10 Âµl 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.
- To image on Phosphoimager (Storm): for 100 ml gel, prepare 250 ml 1x
TAE buffer with 25 Âµl 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.
- Observe gel under UV light and photograph the image. Also, scan the gel under STORM fluorimager.
- 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.
- Observe gel under UV light and photograph the image. Also, scan the gel under STORM Fluorimager to be able to analyze quantitative differences.
Too many bands
Wrong size band
Band in negative RT lane