I am very new to real time PCR (I did my first run today) and i got nothing so i will really appreciate your patience and detailed answers.
As a beginning i wanted to test the expression of one gene only to get used to the technique and to get some results to interpret and understand how the system works, but unfortunately, i got almost nothing.
First i am using power up sybr green master mix and i am doing the run on biorad CfX system.
I put 400 ng of my RNA in the cDNA synthesis in a total reaction volume of 20ul. I assumed a perfect conversion of RNA to cDNA and that i got 400 ng/ 20 ul ( so my conc. is 20ng/ul).
For the syber green, i made a 10 ul total volume that contains 5ul master mix, 1 ul of my cDN (with no dilution = means that i put 20 ng cDNA in the total reaction volume), .5 ul of forward and .5 ul reverse primers (amount of the primers is 500nM) and the rest was water.
Did i made anything wrong in the reaction volumes?
For the set up of the cycling conditions i used the standard cycling recommended by the protocol of power up sybr green. However, the annealing temp i used was 55 C (I knew that people usually use 60 C)
For the dissociation curve, since i was using Biorad machine but power up sybr green protocol, so i could not exactly set up the conditions as the protocol said (the software didn't accept it ) so for example i ended up with a ramp rate of 1.6 c/second instead of .15 C/ sec.
the amplicon of my target gene was around 200 pb in size but my GAPDH control was around 600 pb (i knew that it was away too large :/)
All the results i got were straight lines and only one peak for my control (although i made it duplicate, i saw the peak in only one well)
Could you please advise me what mistake/mistakes i did ? and how exactly to proceed ?
1-Are you sure about the purity of the RNA, did you performed gel electrophoresis for the RNA to detect if the concentration is a pure total RNA or a contamination?
2-Does the cDNA kit expired?, and why you converted 400 ng?. It depends on the type of cells/tissue and the target gene, check out the literatures.
3-I am not expert at molecular biology, but I think 10 ul total volume is not enough, contact BioRad.
4-Optimise the conc. of primers (not the amount) according to the literatures .
5-Optimise the Ann. te,p. by performing gradient step and with gel electrophoresis of the PCR product
Hello Alaa, I have a few ideas for you before you proceed.
First, run your cDNAs on a regular PCR that you usually run and check the gel to see if you have any products, if you get a blank gel that means that your cDNA synthesis did not work.
Second, what standards where in your run? Plasmids or any known positive or negative? That will help you determine where the problem is.
Third, I m not sure of your primer concentration, it seems too high, try diluting them by atleast 1:10, their high concentration may be inhibiting the reaction.
lastly, check how many cycles ran in your experiment, it may have been too low to detect anything
Hello Tara, thank you for your answer
I did a conventional PCR using my cDNA and the gel was perfect. I used +RT, -RT and NTC controls and I didn't get any peaks for any of them. The range of primer conc provided by the master mix protocol is 300-800 nM and I used just 500 nM ! I made 40 cycles as recommended by the master mix protocol!
Hello Ali, thank you for your answer
How could the purity of the RNA affect the real time PCR amplification? I mean I did face some degradation in my RNA. However when I did a cDNA and tested it using conventional PCR it worked great! So I assumed that the degradation issue did not affect the cDNA synthesis.
Of course it's not expired!!!!!!
I converted 400ng / 20 ul reaction volume to get a conc of 20ng/ul
What's the problem with the final volume since all the concerntrations are correct! The 10ul volume is even recommended by the protocol!!!!!
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