The long process from fresh frozen tissue to PCR-ready cDNA seems to me as something quite uncertain. A LOT depends on how well the microdissection goes; and even if you manage to dissect exactly your region of interest, homogenise it properly, the process offers several possibilities of degradation, contamination, mistaked pipetting, and so on.
I am relatively new to this process and well, I feel greatly uncertain about it no matter how careful I am; I'm very unsure about the results. It would be great to hear the opinions and observations of more experienced people.
Thank you for your time!
Hi,
RT PCR has some challenges the good thing is that for many of them you can implement controls. It is important that you have a good quality RNA and comparable quality in all of your samples
Regarding cDNA: In case you have a lot of samples and only limited genes to analyze using a one-Step mastermix might ease the process as it omits the separate cDNA synthesis. In addition (this is for both one-step and two-step) you might consider using an internal control to monitor the RT efficiency. An internal control is a RNA which you can spike into your sample and with this tool you can compare that all samples have the same RT efficiency.
When it comes to the PCR make sure that your PCRs have a good efifciency andcheck that your reference genes ( at least 2 ref genes would be my recommendation) are not affected by your treatment. Finally make sure that you have enough biological replicates and run appropriate controls (e.g. NTC, - RT in case your primers can amplify gDNA, positive control).
Good luck
Sven
Its the gold standard for determining both relative and absolute concentrations of RNA/DNA targets. This allows you to monitor gene expression. It is as robust a tool as western blotting or gel electrophoresis.
Thank you all for taking the time to reply, especially for the details!
I will keep on and be careful, the one-step mastermix sounds like a good way to reduce potential error. I'll read more about the use of internal control!
Dear Teadora
Your could have a look in the Quantinova RT handbooks as they explain the use of the internal control system in case you need more background just let me know and I can provide more by email
Sven
Dear Sven,
Thank you for the suggestion, it was indeed helpful! So internal synthetic RNA control serves as a positive control and makes sure cDNA writing was reliably successful in all cases?
Hi,
in fact this is a tool to control your reverse Transcription and your PCR in general. I would not term it as positive control as it is not a sample for your target of interest
Best Sven
Hi Sven,
I see, thank you for clearing it!
I'm grateful for your help,
Teadora
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