As with many RNA related questions, the answer is it depends. RNAse in inhibitor for your RNA sample is pretty much a must. once it is cDNA then it is not so important.

DNAse treatment is really important if you are using SYBR green and primers that do not span an intron. With primer probe and intron spanning primers it is much less important.

Also if you are using the delta delta Ct method everything is related to a housekeeper. If the expression of your gene is high, and the copy number per genome is low, does it really matter if you measure a bit of gDNA? What accuracy do you need? if you measure 10100 but its actually 10000+ 100 gDNA does that matter?

Treat RNA with DNase

Dear Saeideh,

If you treat your extracted RNA with RNase, you will loose all of your RNA's because RNase digest RNA. And if you treat your RNA extracted samples with DNase, it will digest only the DNA, that might have possibly co-eluted with your RNA samples (which is very common). If you are using the RNA samples for qPCR, then you will have to treat your RNA samples with DNase to digest all the possibly contaminating genomic DNA.

DNAse treatment would be recommended, provided that your RNA isolation method does not support additional means to remove genomic DNA eg. gDNA column in RNesay Plus Mini kit from Qiagen. Using RNase inhibitor in the RT-PCR mix is rather optional but will not hurt your samples.

Treat RNA with DNase and then remove DNase I by Phenol chloroform.

cDNA from RNA I think that the possible answer is treat the RNA with DNAse, so you will get the pure RNA without DNA contamination

Assuming you are doing gene expression studies, treat the RNA with DNase and ensure there is little gDNA contamination by running the RNA samples in a qPCR. Once the samples are free of DNA (very late or no Cq's) you can use the RNA to generate your cDNA. You can also use an RNase inhibitor in the reverse transcription master mix to prevent degradation of your RNA.

I treat my RNA with DNase I, heat-inactivate it (no need to remove it by phenol-chlorophorm) and perform reverse-transcription with and without reverse transcriptase. Use "+ reverse transcriptase" and "- reverse transcriptase" cDNA directly for qPCR. If you don't get signals in your "- reverse transcriptase" qPCR reaction, your reactions are free of DNA contamination.

(Of course, you'll also get no signals in your "- reverse transcripase" if you're using exon-spanning primers even if you've got genomic DNA contamination.)

I treat my RNA samples with DNase.

Treat with DNAse 1 h at 37 °C, heat-inactivated at 65°C in the presence of EDTA for 10 minutes, maker cDNA syntehsis and perform RT_PCR reaction at the first step. The esequence you have to amplify muct conatin intron.

In this caxw in the case of DNA contaminbation you will have 2 bands with small and big size.

In the case of absent of contamination you will have only small band.

DNase treatment will be recommended in this case to avoid gDNA contamination. Moreover, using RNase inhibitor during cDNA synthesis in RT-PCR is absolutely fine. So If you opt both things that will be good. Cheers

as said above we use to treat our material for RNA isolation with potent Rnase inhibitor..and use to treat the RNA with DNAse and further during Reverse Transcription we use RNAse inhibitor enzyme Ribo lock...

I would definitely treat my RNA with DNAse (using the Turbo DNAse kit as Kaushal stated I think it's the best for this work). I would then PCR to see if there is DNA still present. cDNA synthesis was then carried out.

As with many RNA related questions, the answer is it depends. RNAse in inhibitor for your RNA sample is pretty much a must. once it is cDNA then it is not so important.

DNAse treatment is really important if you are using SYBR green and primers that do not span an intron. With primer probe and intron spanning primers it is much less important.

Also if you are using the delta delta Ct method everything is related to a housekeeper. If the expression of your gene is high, and the copy number per genome is low, does it really matter if you measure a bit of gDNA? What accuracy do you need? if you measure 10100 but its actually 10000+ 100 gDNA does that matter?

Many good answers. The best is: it depends. Do you need it DNA free? What is the application? Every additional step will impact your RNA quality. RNAse inhibitor is a must once you want to make cDNA. DNAse treatment is a must if you want to run qRT-PCRs, your primers do not span large introns, and there is no obvious processed pseudogene(s). For qRT-PCR, we design out primers so that there is little chance for amplification from genomic locus of the gene by placing the primers in different exons separated by large introns. If that is not possible, controls are important (as mentioned in previous posts; such as RT negative control)

When I do the extraction RNA, I use DNase I for eliminating possible DNA genomic contamination.Then in the cDNA synthesis, I use a kit with RNAse inhibitor.

As said above I use DNAse during RNA extraction and use even the RNAse inhibitor during cDNA synthesis

Many commercial cDNA kit available with cocktail reagent with RNAse inhibitor, so you can use it. However before cDNA synthesis it is important to treat your RNA prep with DNAse, in my experience, ambio DNase kit is good. Once cDNA is prepared you have to degrade RNA by using RNAse H, which is also part of commercial cDNA kit.

Since it appears that you have censored your question, it may not be answered correctly. However, to make cDNA, the method of choice is to treat with RNAase. However, it depends upon the spanner on your primer.

I would suggest treating the RNA with DNase to remove any contaminating DNA, since even after cDNA synthesis these DNA fragments can linger and can be picked up with RT-PCR.