Yes, you need to treat your RNA samples with DNaseI before cDNA synthesis. 

Hi

It is always preferred to perform this step following the extraction and prior to cDNA synthesis. However, you can skip it if your sure that you primers skip exons.

Wishes,

Bassem

Hi Divyank,

Since most RNA isolates are not entirely free of gDNA, it is indeed always a good idea to treat the isolated RNA with DNAse prior to use for cDNA synthesis and subsequent qPCR. If not done, the gDNA for all genes is still carried over to some extent in your cDNA samples, and this could confound gene expression analyses. But, as Bassem mentions above, (in eukaryotes) if your primers (and/or primers/probe) can be designed to span genomic intron/exon junctions, the DNase treatment is sometimes unnecessary - unless you have pseudogenes involved (which are genomic DNA sequences with introns already removed) - thus which can still be inadvertently amplified if your RNA is left un-DNAsed because you feel your primer designs were "safe".

Back to primers spanning genomic intron/exon junctions:

This maneuver, in other words, means that primers would amplify mRNA only because they are complementary to and inclusive of the exon/exon junction region(s) found only on the mRNA - and thus would only amplify mRNA and not gDNA. Unless your mammalian cells contain pseudogenes for any or all of your targets or reference genes of interest. Mouse GA3PDH (often used as a qPCR reference gene) has a pseudogene associated with it, for instance.

Anna, Bassem, Jack, Thank you so much for those clear and crisp suggestions. 

Could anyone provide me with a DNase removal protocol (Manual) after the DNase Treatment.??

The problem using a kit to remove the residual DNase is that RNA eluted here gets diluted. For eg., if you add 1-2ug of RNA (which is in, say, 2-3ul) for Dnase treatment and purify it using columns, a minimum of 10ul is required to elute it finally which thus dilutes the conc. and therefore makes cDNA preparation a problem (i.e. with 10ul, if one has to use atleast 1ug of RNA) ???

A possible suggestion:

Starting with 1 ug RNA:

If you column elute 10 uL of that RNA, it would be 0.1 ug/uL, then,

Using TURBO DNAse kit:

Add the 10 uL of (0.1 ug/uL) RNA

plus 2 uL 10X TURBO Buffer

plus 3 uL (2U/uL) TURBO DNase

Heat for 30 min at 37C

Add 1.5 uL TURBO Inactivation Reagent

Vortex every 15 seconds for 2 minutes,

Centriguge 3 min at 10,000xg

Remove top 12 uL (avoid bottom portion/Inactivation reagent).

This top 12 uL portion is now ~60.6 ng RNA/uL at this point.

Use 8 uL of this per 20 uL RT rxn.

Assuming high efficiency of conversion to cDNA,

you would obatain ~24 ng/uL cDNA.

Dilute that at least 1:10 pre-qPCR,

 then use 5 uL of this diluted cDNA per each 20 uL qPCReaction.

This will give you ~0.6 ng cDNA/uL in each qPCR - which is plenty for good qPCR.

(If you have the good fortune of being able to start out with more than 1 ug RNA, your situation is even better off than the theoretic scenario above)

Thank you, Jack.