Real Time Quantitative PCR (for Suspension Cells)   

Abstract

Materials and Reagents

1. Suspension cells (such as K562 cells)
2. Chloroform
3. Isopropyl alcohol
4. 75% DEPC-ETOH
5. RNase-free H₂O
6. E.coli RNase H
7. Trizol (Life Technologies, Invitrogen™)
8. Turbo DNA-free kit (Life Technologies, Ambion^®)
9. Superscript III first-strand synthesis supermix (Life Technologies, Invitrogen™)
10. SYBRgreen qPCR Supermix universal (Life Technologies, Invitrogen™)

Equipment

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

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. Add 1 ml of Trizol to lyse cells by repetitive pipetting. Incubate at room temperature for 5 min.
      
   2. 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.
      
   3. Carefully transfer the top aqueous phase (about 0.6 ml) to a new clean tube.
      
   4. (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.
      
   5. Carefully transfer the top aqueous phase to a new clean tube.
      
   6. 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.
      
   7. 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.
      
   8. 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)
   9. 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
      
   10. Add 5 μl Dnase inactivation reagent and mix well.
       
   11. Incubate at room temperature (> 22 °C) for 5 min and mix occasionally.
       
   12. 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)
   13. 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

   14. Gently mix tube contents and incubate at 25 °C for 10 min.
       
   15. Incubate at 50 °C for 30 min.
       
   16. Terminate reaction at 85 °C for 5 min and then chill on ice.
       
   17. Add 1 μl (2 U) of E.coli RNase H and incubate at 37 °C for 20 min.
       
   18. 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)
   19. 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 2x                    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
       
   20. Centrifuge briefly to make sure every component at the bottom of the tube.
       
   21. 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
       
   22. Collect data and analyze the results.

Acknowledgments

This protocol was developed in Len Zon’s lab at Boston Children’s Hospital, Boston, USA, and the work was supported by NIH grant R01 HL04880-21.