Download Q-RT-PCR replaced 4-21-06

©Michael Court
Created Dec 2002
A. DNASE treatment of RNA (to remove DNA contamination)
1. Wear gloves, adhere to RNAse free protocols, and use filter tips
2. Prepare on ice!!
3. In PCR tube:
a. Add 1 ug of total RNA
b. Add 1 uL 10X DNAse buffer
c. Add PCR water to final volume of 9 uL
d. Add 1 uL of DNAse (1 unit; Invitrogen)
e. Mix by pipetting up and down
4. Incubate at room temperature for 15 minutes (do not exceed 15 minutes!).
5. Add 1 uL of 20 mM EDTA (supplied with DNAse) and mix by pipetting
6. Incubate at 650C for 10 min
7. Store on ice (or at -20oC) until use.
B. Reverse transcription of mRNA (prior to QPCR)
1.
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Protocol based on Superscript II (reverse transciptase) – 20 uL
Prepare on ice
In PCR tube:
a. Add 1 ug of total RNA (10 uL from DNAse reaction)
b. Add 1 uL Random hexamer primer (0.1 ug / uL)
c. Add 1 uL dNTP mix (10 mM each)
d. (If needed add PCR water to a volume of 12 uL)
e. Mix by pipetting
f. Brief centrifuge if needed
Put into PCR machine and using program method heat to 650C for 5 min and
cool immediately to 40C.
Brief centrifuge and:
a. Add 4 uL of 5X First Strand Buffer
b. Add 2 uL of 0.1 M DTT
c. Optional --- Add 1 uL Rnase inhibitor (~40 units/uL)
i. [If you don’t use Rnase inhibitor then you must balance with water
to 13 uL at step 3(d) above, instead of 12 uL]
d. Add 1 uL (200 units) Superscript II (reverse transciptase)
e. Mix by pipetting gently up and down with same pipette (change pipettes
between tubes) DO NOT VORTEX
Put into PCR machine and using a program method:
a. Incubate at 250C for 15 min; 420C for 15 min; 500C for 10 min; 600C for
10 min; 700C for 10 min; 40C until use if same day.
b. Add 180 uL PCR water (dilute 10 times).
i. Amount of dilution may vary depending on specific cDNA content
c. Store at -200C for up to 1 month (-800C if longer).
C. Quantitative real-time PCR (SYBR green method)
1. Based on ABI Sybr Green 2X master mix kit and PE 5700 QPCR thermal cycler
2. Thaw cDNA on ice
a. [Return remaining cDNA immediately to freezer once used!]
3. PCR setup can be done at room temperature and stored overnight at 40C
a. DO NOT FREEZE enzyme mix
4. Data should be normalized to a house-keeping gene; currently 18-S-RNA appears
to be the least variable of all genes
a. Need DNAse treatment since no introns in 18SRNA.
5. For each PCR tube prepare the following in a 1.5 mL microcentrifuge tube
(Solution A):
a. 1 uL Forward primer (5 uM; 5 pmole/uL)
b. 1 uL Reverse primer (5 uM; 5 pmole/uL)
c. 0.5 uL PCR water
d. 12.5 uL 2X Sybr green master mix
e. Prepare enough for each tube plus 10 - 20 % more to account for pipetting
waste
f. Mix by pipetting
6. Add 15 uL of Solution A to each PCR tube
a. Use special ABI “Optical” PCR strip tubes and caps
7. To each PCR tube add 10 uL diluted cDNA and mix by pipetting
a. Generally cDNA from RT reaction can be diluted 1/5 to 1/20
b. Dilute enough such that Ct values are >15 but <30 cycles
c. Should include NTC – no template control (10 uL PCR water)
d. Initial evaluation should also include NRTC (no reverse transcriptase
control)
e. Standard curves can be generated using serial dilutions of cDNA with
highest content of mRNA of interest
8. Cap tubes and place in PE 5700 QPCR machine
9. Run at 950C for 10 minutes and then 40 cycles of 950C for 15 sec and 600C for 1
min.
10. Specificity should be checked by:
a. Running a melting curve analysis at end of PCR – should only see a single
sharp peak
b. Running PCR products on a 2% agarose gel – should only see one band
i. BUT Sybr green can cause anomalous motility – so may need to
run separate reaction without Sybr green
c. Purifying and sequencing product