I want to do PCR for the first time. I am a little confused about one step and two step PCR. Which reagent to use? Is there any difference in choosing reagents for one or two step?
Dear Muralidharan, Thank you very much for your reply. Yes i want to do specific gene expression study after drug treatment in a cultured host cell line. Your explanation is certainly helped me to understand the thing.
If I use Qiagen RNeasy mini kit then do I need RLT buffer for cell lysis? and do i need TRIzol in this case when i use RNeasy ?
In real time q-PCR, (supposing you are talking about expression studies in real time) you can go with two kinds of templates, total RNA or cDNA. If you want to use your experimental template as RNA then you protocol will be for one step PCR, conversely if you think RNA may degrade faster and you want to immediately convert it to double stranded DNA and then go for q-PCR then you will use cDNA as your template.
First step- total RNA----------------> cDNA,
Second step- cDNA----------------->amplification plot.
If you want to go from total RNA to amplification plot in one step then you will use RNA as your template in PCR, thats why it is called one step.
If you want to go from cDNA to amplification, then you will have to carry out the first step using reverse transcriptase, and in the second step you will use these double stranded cDNA as template and go for amplification plot, hence called two step PCR.
Coming to the reagents to use:
For cDNA synthesis kit you can use Applied biosystems cDNA synthesis kit or Verso kit from thermo fisher. I use these both.
for the PCR master mix (supposing you want to go for SyBr assay) you can use PowerSyBr green PCR master mix from ABI. Thermo fisher real time PCR master mix if you want to use see if the passive reference dye is included in the master mix. If the ROX dye (passive reference dye) is seperately provided then you will have to optimize your reaction. In short go for the PCR master mix which includes the passive reference dye.
One step qPCR: single tube, RNA->qPCR. Your mix includes RT+qPCR mix.
Two step qPCR: You make cDNA from RNA, then aliquot cDNA into qPCR tube.
One step qPCR is highly convenient, but you have no control over the reaction conditions. You are absolutely committed to the amount of RNA used in that qPCR reaction. Thus, if you are trying to assay many RNA samples which are easily obtained, with few number of genes, then do one step qPCR.
Two step qPCR is more complex, as you have to setup the cDNA reaction. For example, if you use invitrogens superscript III to make cDNA, that is an additional ~2 hours to generate cDNA.
On the other hand, you are able to then use however much cDNA you wish per qPCR reaction. You also have many conditions that you can optimize for the actual cDNA synthesis depending on your targets.
Thus, if you have difficult to obtain RNA samples, have many genes to qPCR, or have difficult/complex qPCR needs, then do two step qPCR.
In my personal experience, unless you have clinical samples or very easy to get samples, two step is the way to go. While it takes setup to do, the headaches it will save downstream when you suddenly realize you want to look at geneX will be worth it, as you thaw that aliquot of cDNA and just run the gene with your original sample.
Cheers and good luck with your qPCR!
*Also, additional source:
http://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2011/09/12/one-step-two-step
Thank you all for your valuable discussion. I hope this will help me indeed. wish you all the best. If I have more query will ask you soon.
Friends, I have another question. Can i see the expression level of more than 1 gene in one PCR experiment? Let say, I want to see the expression of gene A,B,C,D and E (Five genes) I already have the primers for each specific gene. Now, I am going to treat my Cell line with drug and then want to see the expression. So can I use all five primers in one PCR tube after getting the RNA and synthesizing cDNA to see their expression? Or I have to do it separately for five genes? if so, then should I extract the RNA for 5 times of i can use the same extracted RNA/synthesized cDNA and use Primers one after another?
Hope you guys understand my question.
Dear Moyeenul,
As far as i could understand your question is that whether you can check expression for several genes in a single qPCR reaction (known as multiplexing) or you have to perform qPCR for each gene separately (singleplex). If you are going to use intercalating dye based qPCR such as SyBr green, then multiplexing is not suitable but if you are using hydrolysis probe, then it is quite possible but will need a lot of optimization.
hope that helps, good luck
navjyoti
So far your question was about one step and two step. Which is relating RNA or cDNA to be used as template. Now you are asking about singleplexing and multiplexing. If you want to go for single plexing then you have to use Taqman assay and not SyBr green dye based qPCR approach.
Dear Moyeenul,
Since you have a normal PCR in your lab, you would go for reverse transcriptase PCR (rt-PCR). Multiplexing is very much possible but you would need to optimize your primer concentration for each primer very carefully. Also designing of primers will become very critical step. For, rt-PCR and multiplexing I would recommend two-step rt-PCR.
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