REAL-TIME PCR MITOCHONDRIAL DNA DAMAGE ANALYSIS KIT
Human: Cat. # DD2H
Rat: Cat. # DD2R
Mouse: Cat. # DD2M
QUESTION #1:
"Do the QPCR primers contained in the kit bind to the damaged mitochondrial DNA?"
ANSWER:
No. Any damaged DNA or broken DNA can be distinguished from DNA without damage by NO production of 8.8 kb PCR products due to the damaged DNA (see below for the principle of this method).
The mitochondrial DNA (mtDNA) damage kit has 2 components as follows:
Step 1. DNA damage determination:
RT-PCR mtDNA damage analysis kit measures levels of mitochondrial DNA damage by replicating long (8.8 kb for human and 8.2 kb for rat and mouse) mtDNA using QPCR primer mix for replication of 8.8 kb or 8.2 kb pieces. If there is a damage to the DNA, PCR cannot go through. Thus, higher long QPCR product formation represents less DNA damage. Lower QPCR products represent higher DNA damages by adduct formation or cleavage.
Step 2. Quantitation of the 8.8 kb or 8,2 kb QPCR products:
Real-time PCR of the 8.8 kb or 8,2 kb PCR products produced by the Step 1 are carried out using real-time PCR primer mix and the RT-PCR products are calculated using a standard curve obtained using 8.8 kb or 8.2 kb mtDNA standard.
Thus, the 8.8 kb or 8.2 kb mtDNA replication (Step 1) will distinguish DNA damage levels of experimental samples from controls. Then the RT-PCR (Step 2) will replicate the QPCR products to compare DNA damage levels of your experimental samples with the controls.
QUESTION #2:
“Is there any difference between this kit (Cat. # DD2H, DD2R or DD2M) and mitochondrial copy number assay kit (Cat. # MCN1, MCN2 or MCN3)?
ANSWER:
Yes. In the mtDNA damage kit (Cat. # DD2H, DD2R or DD2M), mtDNA damage is measured by carrying out PCR of 8.8 kb or 8.2 kb (long QPCR) and the level of QPCR products will be compared with the level of control mtDNA without damage (Step 1). Quantitation of the 8.8 kb or 8.2 kb QPCR products in Step 2 is carried out by real-time PCR replicating a short fragment (~200 bp).
A chance of a short (~200 bp) DNA to be replicated by RT-PCR is damaged (a bound adduct or broken) is very low (almost nothing) compared to damage on a base in the 8.8 kb or 8.2 kb (8800 bp or 8200 bp) DNA. Long QPCR cannot go through even when a base in the 8.8 kb or 8.2 kb region has an adduct or broken (1 out of 8800 or 8200). After insult, mitochondria in the sample will have the 8.8 kb or 8.2 kb DNA region damaged or not damaged (a mixture of the 2 kinds). The RT-PCR is for quantification of the QPCR products of 8.8 kb or 8.2 kb fragments which didn't have any damage from the insult. The higher level of insult, the lower 8.8 kb or 8.2 kb QPCR products are produced.
In mtDNA copy number measurement (Cat. # MCN1, MCN2 or MCN3), both a short (~200 bp) mtDNA and a short (~200 bp) beta-actin (nuclear DNA) are replicated by real-time PCR and a ratio of mtDNA/nuclear DNA is obtained. High ratio of mtDNA/nuclear DNA means high mitochondrial copy number in a cell.