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Real-time quantitative PCR (qPCR) is an efficient method to detect the levels of gene expression. This protocol provides a complete and detailed procedure for qPCR, including mRNA extraction, genomic DNA removal, cDNA synthesis and targeted gene amplification steps.

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[Bio101] Real Time Quantitative PCR (for Suspension Cells)

Molecular Biology > RNA > qRT-PCR
Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
2/5/2011, 5922 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.27

[Abstract] Real-time quantitative PCR (qPCR) is an efficient method to detect the levels of gene expression. This protocol provides a complete and detailed procedure for qPCR, including mRNA extraction, genomic DNA removal, cDNA synthesis and targeted gene amplification steps.

Materials and Reagents

  1. Trizol (invitrogen)
  2. Turbo DNA-free (Ambion)
  3. Superscript III first-strand synthesis supermix (Invitrogen)
  4. Sybrgreen qPCR supermix (invitrogen)

Equipment

  1. CFX96TM or CFX384TM real-time PCR detection system (Bio-Rad)

Procedure

  1. Total RNA extraction (for suspension cells)
    1. Spin down cells (5~10 X 106) at 1,000 rpm for 5 min at 4 °C.
    2. Add 1 ml of Trizol to lyse cells by repetitive pipetting. Incubate at room temperature for 5 min.
    3. Add 0.2 ml of chloroform and cap tubes securely. Shake tubes vigorously by hand for 15 sec and incubate at room temperature for 2 to 3 min. Centrifuge at 12,000 g for 15 min at 4 °C.
    4. Carefully transfer the top aqueous phase (about 0.6 ml) to a new clean tube.
    5. (Second chloroform extraction) Add 0.6 ml chloroform to the aqueous phase and cap tubes securely. Shake tubes vigorously and incubate at room temperature for 2 min and centrifuge at 12,000 g for 15 min at 4 °C.
    6. Carefully transfer the top aqueous phase to a new clean tube.
    7. Precipitate RNA by adding 0.5 ml isopropyl alcohol to the aqueous phase and mix well. Incubate at room temperature for 10 min. Centrifuge at 12,000 g for 10 min at 4 °C.
    8. Remove supernatant and wash RNA with 1 ml of 75% DEPC-ETOH. Mix the sample by vortexing, and centrifuge at 7,500 g for 5 min at 4 °C.
    9. Remove the supernatant and briefly air dry the RNA pellet ( no more than 10 min). Dissolve RNA in RNase-free H2O by pipetting. Incubate at room temperature for about 5-10 min. Measure RNA concentration using Nanodrop. Check RNA quality by A260/A280 and A260/A230 (RNA with good quality is close to 2 for both). Store RNA at -80 °C.
  2. Remove genomic DNA (TURBO DNA-free kit)
    1. Set up reaction (50 μl)
      RNA prep (up to 10 μg)
      5 μl
      10x buffer
      1 μl
      Turbo DNase
      RNase free H2O
      make up to 50 μl
      Mix gently and incubate at 37 °C for 20-30 min
    2. Add 5 μl Dnase inactivation reagent and mix well.
    3. Incubate at room temperature (> 22 °C) for 5 min and mix occasionally.
    4. Centrifuge at 10,000 g for 1.5 min and transfer the supernatant to a fresh tube. Spec again.
  3. First-strand synthesis (superscript III supermix)
    1. Combine the following kit components in a tube on ice. For multiple reactions, a master mix without RNA may be prepared:
      2x room temperature reaction mix
      10 μl
      Room temperature enzyme mix
      2 μl
      RNA (up to 1 μg)
      X μl
      Nuclease free-H2O
      to 20 μl
    2. Gently mix tube contents and incubate at 25 °C for 10 min.
    3. Incubate at 50 °C for 30 min.
    4. Terminate reaction at 85 °C for 5 min and then chill on ice.
    5. Add 1 μl (2 U) of E.coli RNase H and incubate at 37 °C for 20 min.
    6. Use diluted or undiluted cDNA in qPCR or store at -20 °C until use. (Up to 10% of the qPCR reaction volume may be undiluted cDNA.)
  4. qPCR reaction (sybrgreen qPCR supermix universal)
    1. qPCR reaction (sybrgreen qPaCombine the following components in PCR tube. For multiple reactions, a master mix is strongly recommended to reduce pipetting errors.
      Sybrgreen supermix universal 2 X 10 μl
      Forward Primer (4 μM) 1 μl
      Reverse Primer (4 μM) 1 μl
      cDNA (generated from 1 μg of total RNA) 1 μl
      Nuclease free-H2O to 20 μl
    2. Centrifuge briefly to make sure every component at the bottom of the tube.
    3. Place the reaction in the preheated real-time instrument and program as the following:
      50 °C 2 min (UDG incubation)
      95 °C 10min (UDG inactivation and DNA polymerase activation)
      40 cycles of:
      95 °C 15 sec
      60 °C 60 sec
      Melting curve analysis: refer to instrument documentation
    4. Collect data and analyze the results.CR supermix universal)


How to cite: Jing, L. (2011). Real Time Quantitative PCR (for Suspension Cells). Bio-protocol Bio101: e27. DOI: 10.21769/BioProtoc.27; Full Text



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