Relative RT-PCR: Determination of Primer-to-Competitor Ratio Experiments
Relative RT-PCR: Determination of Primer-to-Competitor Ratio Experiments
The purpose of this scheme is to select an appropriate ratio between the 18SrRNA primer and the competitor that will mimic the expression of the target transcript. For most transcripts, a ratio of 3:7 is typical, and for sparse templates, a ratio of 2:8 or 1:9 may be more appropriate. The PCR reaction mixture should be prepared with 10% more than 5 reactions. This experiment was derived from PCR Laboratory Guide (Second Edition) by Kang Seed and Lijia Qu.
Operation method
Relative RT-PCR: Determination of Primer-to-Competitor Ratio Experiments
Materials and Instruments
Double-distilled water RT-PCR buffer TaqDNA polymerase cDNA dNTP mixture Gene-specific forward primer Gene-specific reverse primer dCTP Move I. Materials For more product details, please visit Aladdin Scientific website.
PCR instrument PCR tubes Quantum RNA Universal 18S standards
1. Buffers, solutions and reagents
Double-distilled water for RNAase removal
10X RT-PCR buffer (100 mmol/LTris-HCl, pH 8.3, 500 mmol/LKC1, 15 mmol/LMgCl2 )
2. Enzyme and enzyme buffer
Taq DNA polymerase (5U/ul)
3. nucleic acids and oligonucleotides
cDNA (from Scheme 1)
dNTP mixture (10 mmol/L, contains all four dNTPs)
Gene-specific forward primers (5 umol/L)
Gene-specific reverse primer (5umol/L)
4. Radiocomplexes
[ a-32P ]dCTP (10uCi/ul) (1Ci=37 GBq)
5. Experimental equipment
Quantum RNA Universal 18S standards (Ambion; includes 18SPCR primer pairs and 18SPCR contenders)
Thin-walled PCR tubes
6. Specialized instruments
Programmable PCR instrument
II. Methods
1.The ratio of primer to competitor is as follows. 
2. Prepare the PCR reaction mixture on ice .
cDNA 5.5ul
10XRT-PCR buffer 27.5ul
10 mmol/LdNTP mixture 22ul
5U/ul TaqDNA polymerase 1.4ul
ddH2O for RNAase removal 177ul
[ a-32P ]dCTP (10uCi/ul) 2.5ul
3. Add 42ul of PCR reaction mixture to each PCR tube on average, 5 tubes in total, labeled 1~5 respectively.
4. Add 2ul of gene-specific forward primer (5umol/L) and 2ul of gene-specific reverse primer (5umol/L) to each of tubes 1~4, respectively.
5. Add a mixture of 18SrRNA primer and competitor as shown below.
Add 4ul of the 1:9 mixture to tube #2.
Add 4ul of 2:8 mixture to tube 3.
4ul of 3:7 mixture in tubes 4 and 5
6. Perform PCR amplification as described in Protocol 1, using the optimal number of cycles determined by the linear range of amplification.
7. Analyze the results of the experiment by denaturing gel electrophoresis as described in Protocol 1. Determine which lane has the amount of 18SrRNA product closest to the expression of the gene-specific product, and the primer-to-competitor ratio of this sample will be used in subsequent relative RT-PCR experiments.