User:Karmella Haynes/Notebook/PcTF Genomics/2014/01/27

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01/27/14

  • qRT-PCR experiment - PlateK562_1/ U2OS
  • U2OS RT-PCR - RNA extraction & cDNA synthesis



RNA Extraction

  • Use combined TRIzol/ spin column metod. Use RNeasy Mini Kit.

Samples (TRIzol-lysed U2OS samples from -80°C freezer)

  1. Tube 1-A (+PcTF)
  2. Tube 1-A (+PcTF)
  3. Tube 4-A (mock)
  4. Tube 4-A (mock)

Procedure notes (based on Carly's notes from 1/10/13)
TRIzol steps

  • Clean all work surfaces and pipettors with RNase-zap.
  • Thaw TRIzol-lysed cells at room temp
  • Under fume hood: Add 100 μL chloroform to each sample
  • Shake vigorously (by inverting tubes) for 20 sec. Let sit at room temp. for 2 min.
  • Centrifuge in cold room (4°C) for 15 min @ 12,000G
  • Pipet off clear phase from organic phase (pink) into new tube (AVOID THE PINK PHASE)
  • Check volume of clear phase (in new tubes). Add same volume of 70% ethanol, RNase-free. Mix and incubate at room temp (refer to RNeasy protocol)

Spin-column steps

  • Open up one pink, sealed spin-column (with attached cap) per sample, label it, and place into collection tube(s).
  • Transfer RNA/ethanol mix to each spin column.
  • Spin tubes at top speed for 30 sec (room temp).
  • Discard flow-through liquid and add 700μL of RW1 Buffer to the spin column.
  • Spin tubes at top speed for 30 sec (room temp).
  • Put collection vial in a fresh collection tube and discard the old one.
  • --> Add 500μL of RBE Buffer to the spin column.
  • Spin tubes at top speed for 30 sec (room temp) then discard the liquid flow-through.
  • Repeat the above two steps again. -->
  • Discard the flow-through and spin again.
  • Transfer liquid to a fresh 1.5 mL tube (RNase-free) and add 30 μL of RNase-free H2O.
  • Let tubes sit for 1 min at room temp.
  • Spin tubes for 1 min (room temp).


Measure RNA Concentration

  • Use the BioTek Take3 plate/ reader
Sample OD 260 260/280 ng/μL
1. 1-A (+PcTF) 1.007 2.1 805.8
2. 1-A (+PcTF) 0.843 2.1 674.0
3. 4-A (+PcTF) 0.992 2.1 793.9
4. 4-A (+PcTF) 0.959 2.1 767.0
  • Keep on ice. Store stock RNA at -80°C after samples are used for cDNA synthesis.


cDNA Synthesis

  • Use Superscript III kit (freezer)
  • Pre-heat the heat block to 65°C. Use the PCR strip-tube-compatible block

Procedure notes

  • Kit components kept on ice: RNase H, SS III RT, and RNase out
  • Kit components thawed at Room temp: Oligo nT, RT Buffer, MgCl2, dNTP mix, water, DTT
  • Label four 0.2 mL PCR strip-tubes
  • Make 8 μL solutions that contain 2.0 μg RNA
    • vol stock RNA (μL) = 2000 ng/ [RNA] from table
    • vol H2O = 8.0 μL - vol stock RNA
  • Make duplicates for each sample. Use RNA stocks #1 and #3.
  1. Reaction 1: 2.5 μL Stock RNA #1 + 5.5 μL H2O = 2.5ug
  2. Reaction 2: 2.5 μL Stock RNA #1 + 5.5 μL H2O = 2.5ug
  3. Reaction 3: 2.5 μL Stock RNA #3 + 5.5 μL H2O = 2.5ug
  4. Reaction 4: 2.5 μL Stock RNA #3 + 5.5 μL H2O = 2.5ug

Part 1

  • Add 1μL of primer (Oligo dT)
  • Add 1μL of dNTP
  • Total volume is 10 μL
  • Incubate for 5 min @ 65°C
  • Immediately place on ice and incubate for 1 min.

Part 2

  • Make a master mix for 4 rxns:
    • Single reaction = 2 μL RT Buffer, 4 μL MgCl2, 2 μL DTT, 1 μL RNaseOut, 1 μL SuperScript III
    • 4 reactions = 8 μL RT Buffer, 16 μL MgCl2, 8 μL DTT, 4 μL RNaseOut, and 4 μL SuperScript III
  • Carefully add 10μL of MM to each rxn tube
  • Mix tubes gently by flicking, centrifuge for a few seconds

Part 3

  • RT-PCR Machine, set up as follows:
  1. Stage 1: 50 min @ 50°C
  2. Stage 2: 5 min @ 85°C
  3. Stage 3: Infinity @ 4°C
  • Add 1μL: of RNase H
  • Incubate @ 37°C for 20 min
  • Store at -20°C

qRT-PCR - Plate K562_1/ U2OS


  Template Gene Target
Rxn 1: treated cells KIT, primers 13/14
Rxn 2: treated cells TNFRSF11A, primers 15/16
Rxn 3: treated cells EGFR, primers 17/18
Rxn 4: treated cells WT1, primers 19/20
Rxn 5: treated cells HLF, primer 21/22
Rxn 6: treated cells BCL6, primer 23/24
Rxn 7: treated cells mCh
Rxn 8: treated cells ref. gene, GAPD
Rxn 9: untreated cells KIT, primers 13/14
Rxn 10: untreated cells TNFRSF11A, primers 15/16
Rxn 11: untreated cells EGFR, primers 17/18
Rxn 12: untreated cells WT1, primers 19/20
Rxn 13: untreated cells HLF, primer 21/22
Rxn 14: untreated cells BCL6, primer 23/24
Rxn 15: untreated cells mCh
Rxn 16: untreated cells ref. gene, GAPD
Rxn 17: no template KIT, primers 13/14
Rxn 18: no template TNFRSF11A, primers 15/16
Rxn 19: no template EGFR, primers 17/18
Rxn 20: no template WT1, primers 19/20
Rxn 21: no template HLF, primer 21/22
Rxn 22: no template BCL6, primer 23/24
Rxn 23: no template mCh
Rxn 24: no template ref. gene, GAPD


Primer/Probe Master Mixes (8 total)

Reagent (Single well) Gene Target 1 - 7 (x10) Gene Target GAPD (x10)
2x LC480 Probes Master (7.5 μL) 75 75
20 μM Forward primer (0.3 μL) 3 3.0 GAPD primers*
20 μM Reverse primer (0.3 μL) 3 ---
10 μM UPL probe (0.3 μL) 3 3.0 GAPD UPL probe*
PCR H2O (0.1 μL) 1 4
Total vol. (8.5 μL) 85 85


Template Master Mixes

  • Use 1:10 dilution of cDNA for targets
  • Use 1:100 dilution of cDNA for GAPD
Reagent (Single well) treated cDNA Template (x25) untreated cDNA Template (x25) no Template (x25)
diluted cDNA (2.0 μL) 50 50 ---
PCR H2O (4.5 μL) 112.5 112.5 162.5
Total vol. (6.5 μL) 162.5 162.5 162.5


96-well plate

  • First of each triplicate well, add 25.5 Primer/Probe MM + 19.5 Template MM
  • Transfer 15 μL from well 1 to wells 2 and 3 for each set