I am worried about the concentration of cDNA used in PCR normal. Is it to be directly used to compare gene overexpression and downregulation? Some people are addicted to do direct PCR from 3ug cDNA which is really high but the master mix they use was previously used for PCR of cDNA with concentration of 100ng cDNA so alost 30 times high concentration of cDNA. How one can draw an analysis from that?
Is it a right method to do so?
When you say "normal" PCR reactions are you referring to RT-PCR or qPCR? When you are trying to compare upregulation or downregulation of a gene using qPCR, whether it be SYBR or TaqMan assay, you do not need 3 ug. You can most certainly use 100 ng.
In my experience though usually when I say I am using "100 ng" I am referring to the input RNA used to make cDNA. As long as you have the same input RNA in your cDNA reaction then you can just use equal volumes for your qPCR reaction. The cDNA kits I have used have things in the buffer that prevent using a nanodrop to read concentration anyways.
If you could provide more specific information about what type of PCR you are doing that would probably get you a better answer.
I get used to dilute my cDNA samples 1 : 5 (cDNA : water) before using it to RT-PCR or normal PCR (for example to get the gene for cloning), and it worked perfectly with me.
Try that and let us know, Good Luck
for RT-PCR 50ng of cDNA is sufficient u may use the same or less for quantitative RTPCR
Hi Bhavin
One usually does a standard curve with cDNA of known concentration. You then also dilute your cDNA (1:5 or 10) should suffice. When run against the standard curve (which should be included in each experiment according to MIQE guidelines necessary for easier QC and publication) you should have a good idea of whether you should dilute cDNA more or less for further experiments.
Good luck
You need only do a standard curve if you are looking for absolute quantization. Otherwise, if you are doing a relative quantitization and normalizing to a housekeeping gene you don't need a standard curve.
Thanks a lot for your time and suggestion to my question. We do isolate RNA from fly and take 3ug and then make cDNA. We dilute cDNA to make it 100ng/ul and then do normal PCR reaction (no Q PCR). We are supposed to follow the same but few of my colleagues use direct cDNA without normalizing or diluting to a certain concentration. According to me its malpractice but i asked to clarify is it right or not??? So many have answered it as a wrong. We check over and downregulation of gene so is it a right protocol???? I don't think so and that is why I put this question on forum ....If you have paper available please send it to me on my id "[email protected]" Thanks in advance....
Hello everyone !!!! No one has added an answer to my question or suggestion then. Please do send me ideal protocol for normal PCR from cDNA or paper which I can use for my experiments.
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