Relative RT-PCR: Sample quantification assay
Relative RT-PCR: Sample quantification assay
Knowing the linear range of cycling parameters and the ratio of 18SrRNA primer to competitor, one can use one's own samples for relative quantitative RT-PCR.The PCR reaction mixture formulated below contains 9 reactions (4 samples, 4 non-reverse-transcription controls, and 1 control with no template added) and an additional 10% share.The PCR reaction mixes below contain 9 reactions (4 samples, 4 non-reverse-transcription controls, and 1 control without template) and 10% extra. This experiment was derived from PCR Laboratory Guide (2nd edition) by Kang Seed and Lijia Qu.
Operation method
Relative RT-PCR: Sample quantification assay
Materials and Instruments
Double-distilled water RT-PCR buffer MMLV Reverse transcriptase SUPERaseINRNAase inhibitor Taq DNA polymerase Total RNA Random primers dNTP mixtures Gene-specific forward primers Gene-specific reverse primers [a-32P]dCTP Move I. Materials For more product details, please visit Aladdin Scientific website.
Quantu mRNA Universal 18S standards PCR Tube PCR Instrument
1. Buffers, solutions and reagents
Double-distilled water for RNAase removal
10X RT-PCR buffer (100 mmol/L Tris-HCl, pH 8.3, 500 mmol/L KCl, 15 mmol/L MgCl2 )
2. Enzyme and enzyme buffer
MMLV reverse transcriptase (100-200 U/ul)
SUPERaseINRNA enzyme inhibitor (20U/ul;Ambion)
Taq DNA polymerase (5U/ul)
3. Nucleic acids and oligonucleotides
Total RNA (up to 2.5ug per sample)
Random primers (50umol/L)
dNTP mixture (10 mmol/L, contains all four dNTPs)
Gene-specific forward primer (5 umol/L)
Gene-specific reverse primer (5umol/L)
4. Radiocomplexes
[ a-32P ]dCTP (10uCi/ul) (lCi=37 GBq)
5. Experimental equipment
Quantu mRNA Universal 18S standards (Ambion; includes 18S PCR primer pairs and 18S PCR contenders)
Thin-walled PCR tubes
Specialized instruments
Programmable PCR Instruments
II. Methods
1. Run the single-stranded cDNA synthesis reaction centrally as described in Scheme 1, using the same amount of RNA for determining the linear range of all Ben's products.
2. Prepare the PCR reaction mixture on ice.
10XRT-PCR buffer 50ul
10 mmol/LdNTP mixture 40ul
5umol/L gene-specific forward primer 20ul
5umol/L gene-specific reverse primer 20ul
18S rRNA primer and competitor (added at the ratio determined by protocol 2) 40ul
5U/ul Taq DNA polymerase rk; 2.5ul
[a-32P]dCTP (10uCi/Hl) 5ul
Double-distilled water for RNAase removal 312.5ul
3. Add 49ul of PCR reaction mixture to each of the 9 thin-walled PCR tubes.
4. Add 1ul of ddH20 to the control tube without template.
5. Add 1ul of each sample RNA to the corresponding non-reverse transcription control tube.
6. Add 1ul of each sample cDNA from the first cDNA synthesis step to the appropriate tube.
7. Perform PCR, as described in Protocol 1, using the optimal number of cycles as determined by the linear range of amplification.
8. analyze the results of the experiments by denaturing limb electrophoresis as described in Scheme 1. Controls without template and non-reverse transcription controls should produce no PCR products. The relative amount of PCR product in a sample can be determined by dividing the signal of the gene-specific product by the signal of the 18S rRNA, thus obtaining an accurate relative value for the gene-specific product in each sample, which can be used to compare the relative expression of template RNA between samples. Using the normalized signal of Sample 1 / normalized signal of Sample 2 yields a ratio expressed as a multiple or percentage. An example of a relative quantitative RT-PCR is shown in Figure 13-4. 