Mouse/Rat Pluripotent Stem Cell Assessment Primer Pair Panel

Assay Procedure

Refer to the product datasheet for complete product details.

Reagents Supplied in the Mouse/Rat Pluripotent Stem Cell Assessment Primer Pair Panel (Catalog # SC015)

• Double stranded DNA to serve as a positive control
• 14 primers to amplify stem cell markers
• 1 primer to control for cDNA synthesis (GAPDH)

Reagents

• RNase-free DNase (DNase I)
• Random Primers (Catalog # RDPC2) or Oligo (dT)12-18 Primers (Catalog # RDPC1)
• RNase H- Reverse Transcriptase
• Taq DNA Polymerase
• Agarose
• Reverse Transcription Buffer
• 10X Taq Buffer
• 2 mM dNTPs
• 10 mM dNTPs
• 0.1 M DTT
• Autoclaved Deionized Water
• Nuclease-free Water
• Gel Loading Dye
• DNA Markers
• Mineral Oil
• 0.1X TE
• 1X TAE

Materials

• Pipettes and Aerosol Barrier Pipette Tips
• Gloves

Equipment

• Thermal Cycler
• Hot Block or Water Bath at 70 °C
• Hot Block or Water Bath at 42 °C
• Thin Walled PCR Tubes
• 1.5 ml Sterile Tubes

Specificity

All mouse/rat pluripotent stem cell primer pairs are specific for mouse and rat cDNA. All primer pairs were also tested on human cDNA. The GATA-4 primer pair amplified human cDNA. The mouse/rat GATA-4 primer pair is not optimized for human. All pluripotent stem cell primer pairs were tested with mouse embryonic fibroblast (MEF) cDNA and 13 of the 14 primer pairs did not amplify a product. Mouse/rat Nestin did amplify a product from MEF cDNA.

I. DNase Treatment

1. To avoid false positives, contaminating genomic DNA must be removed by treatment of total RNA with RNase-free DNase. Follow the procedure according to the manufacturer’s instructions.

II. Reverse Transcription Reaction

1. Thaw all reagents completely on ice. All reactions should be assembled on ice.
2. Pipette the following into a nuclease-free tube:
   • 1-5 μg of DNase treated total RNA (up to 11 μL)
   • 1 μL of random primers (300 ng/μL) or Oligo (dT)_12-18 primers (0.5 mg/μL)
   • X μL of nuclease-free dH₂O for a final volume of 12 μL
3. Mix and incubate at 70 °C for 10 minutes and then place tubes on ice immediately.
4. Briefly centrifuge the tube and add the following to each tube:
   • 4 μL of 5X Reverse Transcription Buffer
   • 2 μL of 0.1 M DTT
   • 1 μL of 10 mM dNTPs
5. Mix and incubate at room temperature for 10 minutes.
6. Incubate at 42 °C for 2 minutes.
7. Add 1 μL of RNase H- Reverse Transcriptase (200 units/μL). Mix by pipetting.
8. Incubate at 42 °C for 50 minutes.
9. Incubate at 70 °C for 15 minutes.
10. Dilute reactions 5-fold by adding 80 μL of nuclease-free dH₂O.
11. Assay immediately or store the cDNA samples at < -20 °C in a manual defrost freezer.

III. PCR

1. Resuspend each primer pair in 50 μL of autoclaved deionized water or 0.1X TE Buffer (1 mM Tris HCl, 0.1 mM EDTA, pH 8.0 at 25 °C) for a final concentration of 7.5 pmoles/μL each primer. Do not resuspend the Positive Control at this time.
2. Determine the number of PCR reactions (see Technical Hints for setting up a PCR master mix). Multiply the volumes listed below for each reagent by the number of reactions. Prepare a separate master mix for each primer pair.
   • 36.5 μL autoclaved deionized water
   • 5 μL 10X Taq buffer (with 15 mM MgCl₂)
   • 5 μL 10X dNTP mix (10X= 2 mM each dNTP)
   • 2 μL Primers (7.5 pmoles/μL each primer)
   • 0.5 μL Taq DNA Polymerase (5 units/μL)
3. Prepare the Negative Control reaction tube(s):
   1. Pipette 1 μL of autoclaved deionized water into a pre-labeled negative control tube.
   2. Add 49 μL of the master mix prepared in step 2.
   3. Briefly spin the tube.
   4. Add 30 μL of mineral oil to prevent evaporation.
   5. Close the reaction tube and place on ice.
4. Prepare the cDNA sample reaction tubes:
   1. Pipette 1 μL of cDNA sample into a pre-labeled PCR reaction tube.
   2. Add 49 μL of the master mix prepared in step 2.
   3. Briefly spin the tube.
   4. Add 30 μL of mineral oil to each tube to prevent evaporation.
   5. Close the reaction tube and place on ice.
5. Resuspend the Positive Control 57 in 150 μL of autoclaved deionized water or 0.1X TE Buffer. Centrifuge the Positive Control tube briefly. This should be done in a separate location from where the PCR reactions are set up. Use different pipettes than those used for PCR set up.
   1. Pipette 1 μL of Positive Control into the pre-labeled Positive Control reaction tube.
   2. Add 49 μL of the master mix prepared in step 2.
   3. Briefly spin the tube.
   4. Add 30 μL of mineral oil to the tube to prevent evaporation.
   5. Close the reaction tube and place on ice.
6. Place all tubes in a thermal cycler and use the following PCR program:
   • 94 °C for 4 minutes
   • 30-35 cycles:
     • 94 °C for 45 seconds
     • 55 °C for 45 seconds
     • 72 °C for 45 seconds
   • 72 °C for 10 minutes

IV. Run PCR Products on Agarose Gel

The PCR products can be analyzed by 1.5-2% agarose gel electrophoresis. For predicted sizes of PCR products, please refer to Table 2 in the datasheet.