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Protocols and Guides

qPCR Protocol :

Materials Needed:

1. DNA or RNA samples
2. qPCR Master Mix (commercially available, includes DNA polymerase, dNTPs, buffer, etc.)
3. Primers specific to the target gene
4. Probe (if using TaqMan or similar methods)
5. Reverse transcriptase (for RNA samples, if performing RT-qPCR)
6. PCR tubes or plates
7. Real-time PCR machine (thermal cycler)
8. Pipettes and tips
9. Nuclease-free water

Protocol Steps:

1. Prepare the Reaction Mix:
   • For DNA samples:
     • Prepare a reaction mix containing the following per reaction:
       • qPCR Master Mix: 10 µL
       • Forward Primer (10 µM): 0.5 µL
       • Reverse Primer (10 µM): 0.5 µL
       • Nuclease-free water: 8.5 µL
       • DNA sample: 1 µL (template DNA)
   • For RNA samples (RT-qPCR):
     • First, convert RNA to cDNA using a reverse transcription step:
       • RNA (variable amount, typically 1 µg per reaction)
       • Reverse transcriptase enzyme
       • Random primers or oligo-dT primers
       • Incubate according to the manufacturer’s guidelines.
     • After cDNA synthesis, use the following for qPCR:
       • qPCR Master Mix: 10 µL
       • Forward Primer (10 µM): 0.5 µL
       • Reverse Primer (10 µM): 0.5 µL
       • Nuclease-free water: 8.5 µL
       • cDNA sample: 1 µL (template cDNA)
2. Mix the Reaction Components:
   • Combine the master mix, primers, and water carefully.
   • Add the template (either DNA or cDNA).
   • Gently pipette up and down to mix, but avoid bubbles.
   • Centrifuge briefly to collect any liquid from the sides of the tubes.
3. Prepare the qPCR Plate/Tubes:
   • Load the reaction mixture into the wells of a qPCR plate or into PCR tubes, ensuring each well/tube receives the correct volume of reaction mix.
   • Seal the plate or cap the tubes securely.
4. Set Up the qPCR Machine:
   • Load the prepared plate or tubes into the real-time PCR machine.
   • Set up the cycling conditions on the thermal cycler:
     • Initial Denaturation: 95°C for 3-5 minutes (to activate the polymerase and denature the DNA).
     • Amplification Cycles (40 cycles):
       • Denaturation: 95°C for 15 seconds
       • Annealing: 55-65°C for 30 seconds (depends on primer Tm)
       • Extension: 72°C for 30 seconds
     • Detection: Measure fluorescence at the end of the extension phase.
   • If using a probe-based method (e.g., TaqMan), make sure to include the correct detection settings for the fluorophore in use.
5. Run the PCR Program:
   • Start the qPCR machine and allow it to complete the run.
   • The real-time PCR machine will record the fluorescence at each cycle, which correlates with the amount of DNA or cDNA in each sample.
6. Analyze the Data:
   • After the qPCR is complete, the machine will generate an amplification curve for each sample.
   • The point at which fluorescence exceeds the threshold (Ct or cycle threshold) is used to quantify the amount of target DNA or RNA.
   • Use the standard curve method (for absolute quantification) or the ΔCt method (for relative quantification) to analyze the data.
7. Post-PCR Analysis:
   • Review the amplification curves for consistency.
   • Check for any primer-dimer formation or non-specific amplification by examining the melting curve (if your qPCR machine has this capability).

Tips for Successful qPCR:

• Ensure the primers are specific to your target gene to avoid non-specific binding.
• Use high-quality DNA or RNA samples, free of contaminants that could interfere with PCR.
• For RNA samples, always include a no-reverse transcriptase control (to check for genomic DNA contamination).
• Perform technical replicates to ensure the reliability of your results.
• Always include a negative control (no template) to detect contamination.