Villanueva, Laura 1-5
Quantitative / Real Time PCR
1.
Design sets of forward & reverse primers a.
Use Primer Express or Primer3 i.
http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi
2.
Primer design rules: a.
Amplicons should be 50-150 bp, definitely not more than 200 bp b.
Primer length is 15–25 bp, optimally 20 bases c.
Primer GC content 30-80% d.
Primer melting temperature (Tm) of 58-60 o
C
3.
Check the the last five nucleotides at the 3' end of each primer a.
Good primers should have no more than 2 G and/or C
4.
Double-check the good primers with NetPrimer a.
www.premierbiosoft.com/netprimer/index.html b.
Check for secondary structure i.
One hairpin or one dimer is ok but not both. c.
Check for runs i.
Avoid runs of three or more identical nucleotides. ii.
One run of 3 A or 3 T is ok, but not 3 G or 3 C iii.
Ideally not more than one run d.
A score for the quality of your primer is given i.
A score of 100 is ideal.
5.
Order 3 or 4 sets of primers
6.
Test the primer sets with DNA and use the best pair for QRT-PCR.
7.
Run a gradient PCR with different template and salt concentrations. a.
10
 l Q solution (Qiagen) b.
3
 l MgCl
2
(25 mM) c.
5
 l 10X Buffer d.
4
 l dNTPs (0.25 mM) e.
2.5
 l Forward primer (4 pmol/
 l) f.
2.5
 l Reverse primer (4 pmol/
 l) g.
1
 l BSA 100X h.
21.5
 l water for a 50
 l final volume. i.
It’s better to use less concentration of primers if you are amplifying small bands as an excess of primers can interfere the visualization of your band in the gel
8.
Look for one band in the PCR product with no unspecific hybridization/bands.
9.
Join several PCR products
10.
Gel purify the band a.
Use GenCatch gel extraction kit from Epoch Biolabs, MinElute Gel
Extraction Kit from Qiagen or QiaQuick gel extraction kit from Qiagen. b.
After gel extraction purification, join DNA aliquots, dry down in Speed
Vac and resuspend in less volume. c.
Check concentration and purity in Nanodrop. d.
Final DNA yield should be more than 30 to 40 ng/
 l. e.
Use this DNA for the standard curve.

Villanueva, Laura 2-5
11.
Clone the purified product
12.
Sequence the product to check if the cloned sequence is the right one.
13.
DNA Standard curve a.
Make a 1:10 dilution of a 200 pmol/
 l stock primer or a 1:2 dilution of a
40 pmol/
 l stock primer b.
Measure the A
260 nm
in the Nanodrop. c.
Enter the values in the “Taqman file” excel file to get the volume of the primer stock that has to be pipeted to get a 0.15

M concentration in 500
 l final volume. d.
Enter the size of the amplicon, position of the primers and the concentration of the purified DNA obtained by Nanodrop in the “Taqman” file and you’ll get the molecules of DNA per  l of sample. e.
DNA dilutions: i.
Starting from the purified DNA stock, make the first dilution 1:10
(4
 l DNA + 36
 l MQ water), pipette up and down and make the rest of the dilutions –10 to –4
24.69 ng/
 l molecules per
 l l
As we add 9.5
’re going to
 l in the real there ’s 2.1e+11 f.
Set up the standard DNA curve i.
Wear gloves always! ii.
Keep on ice at all times! iii.
Pipette everything a lot! iv.
Make a mix with the primers and SYBR green for each reaction:
1.
1.5
 l Forward primer (0.15

M)
2.
1.5
 l Reverse primer (0.15

M)
3.
12.5
 l Power SYBR green PCR master mix. v.
Considering at least 3 reactions more then needed for a master mix. vi.
Add 9.5
 l DNA of the specific dilution
1.
All dilutions have to be set up in triplicate in a 96-well optical plate (Applied Biosystems).
2.
Avoid bubbles especially at the bottom of the tubes (the plate can also be spun down). vii.
Seal the plate with the optical adhesive avoiding the formation of bubbles.

Villanueva, Laura 3-5 viii.
Keep at 4

C. ix.
Run the plate in the RT-PCR system:
Make sure the computer is not connected to the network before starting.
Turn on the system and then the computer.
Open the program 7500 system software icon.
Open a new document
Next (Absolute quantification)
Select to create a new detector o Reporter: SYBR o Quencher: None o Passive reference: ROX
Create different detectors if you want to test more than one set of primers in the same plate (one per each set of primers).
Next and Finish.
Filling in the plate: o Click onto the squares. o Name the sample o Select the detector o Select the task (Standard or Unknown) o Close. o Remember that if you are entering the standard samples you have to indicate in the “Quantity” the number of molecules of DNA per well.
Save.
1 2 3 4 5 6 7 8 9 10 11
-10
-6
-10 -10
-6 -6
-9
-5
-9
-5
-9
-5
-8
-4
-8
-4
-8
-4
-7 -7 A
B
C
D
E
F
G
H
Instrument: Thermal profile. o You can leave everything by default o Increase the repetitions of the stage 3 to 50. o If you are testing primers and the standard curve for the first time add an extra dissociation stage to check that your primers are good.

Stage 4:

1 Rep
 95ºC 15 secs
 60ºC 1 min
 95ºC 15 secs o Change volume of the reaction to 25
 l o Save again.
12
-7

Villanueva, Laura 4-5
Insert the plate in the system
Close the compartment
Click on Start.
Runs for approximately 2 hours 20 minutes.
Analyze the results: o Select the wells of the standard curve and click on the arrow. o Go to the Delta Rn vs Cycle curve and set the threshold manually in the exponential phase of the amplification curve o See the Power Sybr Green guide. o Analyze starting from cycle 1 to 2. o Go to the standard curve: o The “perfect” standard curve should have a correlation coefficient close to 1 and a slope around -3.300. o If you see that some of your points are giving problems to adjust the curve, you can go to the plate representation, select the wells and omit them in the upper bar menu. o Select all the wells of the standard curve again including those omitted and click arrow again. o Save.
Check the Melt curve. o The melt curve is a good indication of how clean the PCR product amplified is; ideally a single narrow peak.
Choose the set of primers that give the best DNA standard curve and also that generate the best melting curve.
14.
Once you get your RNA, you have to check the following things: a.
Good ratio A
260
/A
280
≥1.8 b.
Run an agarose gel. i.
23S/16S rRNA ratio should be 2:1 ii.
No degradation should be observed. c.
Set up a PCR reaction with your original primers and the RNA after the
DNase treatment. i.
No amplification bands should show no or low DNA contamination
15.
Reverse transcription:
16.
Perform the reverse transcription with the proper negative controls. a.
For each gene/set of primers 1 st
tube:
Tube Water dNTPs Primer RNA
A (+)
B (-)
1
1
2
2
2
2
5
5
C (-RNA) 6 2
Quantities in microliters.
2 -
Water and dNTPs provided by the Enhanced Avian HS RT-PCR kit from Sigma.
Primer: Reverse primer 20 pmol/
 l.

Villanueva, Laura 5-5
RNA: Use 5
 l of your purified RNA with a minimum concentration of about 30 ng/
 l.
17.
Confirm the conversion to cDNA by PCR amplification with the selected primers.
18.
Run a Real Time-PCR plate with the sample to be quantified and the standard
DNA dilutions in triplicate.
19.
Follow the same protocol for setting everything up as used with the standard curve.
20.
Instrument Thermal profile for a reaction with the standard curve and the
Unknown samples:
Stage 1: o Reps 1 o 50ºC 2 min
Stage 2: o Reps 1 o 95ºC 10 min
Stage 3: o Reps 50 o 95ºC 15 secs o Tm 1 min
-
The melting temperature by default is 60ºC and is a melting and extension temperature at the same time.
However, if you’re having problems amplifying your target you can set your Tm for 45 secs and then add a 72ºC step for 35 secs.
21.
Analyze the data
22.
If it looks fine run again with the sample in duplicate.
IMPORTANT CONSIDERATIONS:
We have observed that there are some little things that can make a difference in your
Real-time analysis:

Primer stock: o Make some 0.15 μM primer aliquots for the real time reaction at the same time and keep them at −20ºC. o Don’t use an aliquot more than once (there’s a decrease in the efficiency).

Power SybrGreen kit: o The kit can be stored at −20ºC, but once you use it should be keep in the fridge.

Standard DNA dilutions: o Better results if you mix the SybrGreen + primers mix and the appropriate amount of DNA for the 3 replicates and then dispense them in the plate.

Unknown samples: o Pipette the reverse transcription product in the well before adding the
SybrGreen + primer mix. o Don’t pipette up and down. o Just spin the plate 1000 rpm for 15 sec.