Real-time PCR

I. Primer design using Vector Nti (Adopted from Matt)
Note: Primer design software in DNASTAR also works, as long as the primers meet the same criteria as indicated below.

a. Obtain the cDNA sequence of your gene of interest from GenBank and save it in Vector NTi 7.0 database.

b. Highlight the 3’UTR region of the gene and search for oligos by choosing “PCR Primers” under “Analyze”. (If it is a small gene, you could use the whole gene sequence to choose primers. However, 3’ UTR is a better choice, since there is much less cross-hybridization between species in this region.)

c. Under “Primer”, choose amplicon length 75-150 (this length is optimal for real-time PCR, giving an efficient reaction. If it is impossible to design good primers within this length, one could try slighly longer length.). Tm(c) is 50-65, %GC is 50-60, and primer length is 17-20.

d. Under “Structure”, set Nucleotide Repeat to “Max 3”.

e. Under “3’ end”, uncheck the A and T in the 1st Nucleotide category for both sense and antisense primers.

f. Under “Quality” field and gives high priority (10) to the Tm and GC content.

g. Click OK and look at the list of oligos generated. Discard those that have any of the following characteristics:

  • Have As or Ts on the 5’ ends of primers.
  • Primers that have a stretch of Gs or Cs have lower priority.
h. Once a likely set of primers has been identified, copy the sequences to the Operon website (www.operon.com/oligos/toolkit.php) and check the Tm and sequences of both primers for possible primer-dimer formation. Also check for any 3’ complimentarity between the primers (both with themselves and with each other). This area is particularly important in the formation of primer-dimers. In addition, minimize the stretches of internal complimentarity to less than 4 bases in length.

i. Check primer folding at Dr. Zuker’s web site (bioinfo.math.rpi.edu/~mfold/dna/form1.cgi).
On the form, following these instructions:
  1. Give the sequence a name.
  2. Copy or type the sequence in the big box.
  3. Go to the field marked “ionic conditions” and change the units to mM. Set Na to 50 and Mg to 3.
  4. Enter a temperature around 2-3 degrees below the annealing temperature of your reaction (55-60oC).
  5. Go to the email field and enter your email address (nothing will be mailed to you, though).
  6. Click the fold DNA button.
The program will give you a list of structures. Check the ΔG values. Any structures with positive values can be ignored. If the value is negative, check its Tm. If the Tm is high (similar or above the Tm of primer), the primer should not be used.

j. Do a Blast search (www.ncbi.nlm.nih.gov/blast) against est_mouse. The primers should not have perfect match in the mouse EST database.

II. cDNA templates preparation
Reverse Transcription

I use Taqman Reverse Transcription Reagents (Applied Biosystems, # N8080234, $280.50) because the cDNA product can be directly used for the following PCR reaction without further purification. The total RNA template should have been treated with RNase-free DNase during or after its preparation, followed by purification with Rneasy kit.

A template for the reaction mix is shown below.

RT Reaction for Real-time PCR
 
Sample Name            
Conc. Of RNA            
10x RT 10 10 10 10 10 10
25 mM MgC12 22 22 22 22 22 22
dNTP Mix 20 20 20 20 20 20
Oligo (dT) 16 5 5 5 5 5 5
Rnase Inhibitor 2 2 2 2 2 2
Reverese Trans. 2.5 2.5 2.5 2.5 2.5 2.5
RNA (2ug)            
H2O (38.5-RNA)            
Total (ul) 100 100 100 100 100 100
Thermacycling conditions for RT:

Step 1: 25oC 10 min.
Step 2: 48oC 30 min.
Step 3: 95oC 5 min.

III. PCR using icycler from Bio-Rad
(Turn on the iCycler, camera, and the computer consecutively about 30 min before the reaction.)
Use the above RT mix directly as the template for PCR. For the first time of using a primer pair, one should always do a standard curve. A series of dilutions of the cDNA sample are used as the templates, and the iCyler software will automatically draw a standard curve based on the PCR result. Theoretically the coefficient of the curve should be >0.99, although some primer pairs with a coefficent of 0.97 also worked in our hands. The slope of the curve should be as close to 3.32 as possible. For RNA samples that might be contaminated by mouse RNA, a separate PCR using mouse cDNA should be performed and no or very little product should be detected.

Finally, you are ready to do the real Real-Time PCR. Here is a template of making the reaction mix in a 96-well plate (#2239441, Bio-Rad).
REAL-TIME PCR SHEET
(µl)
WELLS SYBRPCR Master MIX (#4309155 from Applid Biosystems) 5µM upstream primer 5µM downstream primer cDNA Template Dilutions H2O Total
               
A1-A3 25 3 3 5   14 50
A4-A6 25 3 3 5   14 50
A7-A9 25 3 3 5   14 50
A10-A12 25 3 3 5   14 50
B1-B3 25 3 3 5   14 50
B4-B6 25 3 3 5   14 50
B7-B9 25 3 3 5   14 50
B10-B12 25 3 3 5   14 50
C1-C3 25 3 3 5   14 50
C4-C6 25 3 3 5   14 50
C7-C9 25 3 3 5   14 50
C10-C12 25 3 3 5   14 50
D1-D3 25 3 3 5   14 50
D4-D6 25 3 3 5   14 50
D7-D9 25 3 3 5   14 50
D10-D12 25 3 3 5   14 50
E1-E3 25 3 3 5   14 50
Note: I usually make a master mix of everything except the cDNA templates for each primer pair, and use multi-pipettors to aliquote into each well. The cDNA templates are added to each well individually.

After aliquoting the reaction mix, the plate should be sealed with an iCycler Optical Quality Sealing Tape (#223-9444, Bio-Rad). Only touch the edge of the tape! Any smear on the part that covers samples will affect results. Smooth the tape using a roller. Sit the plate on the plate holder in iCycler. Make sure the plate is not tilted.

Set thermacycling conditions for PCR in icycler (under “Protocols”):

Step 1: 95oC 10 min.
Step 2: 95oC 15 sec.
Step 3: 60oC 1 min.
40 cycles between Step 2 and Step 3.

Check the plate set up. Highlight all the samples with green marker.

When everything is all set, click on the “Run” button.