I use 1ug of total RNA in cDNA reaction using revert aid first starnd cDNA synthesis
then I use 1ul in qpcr reaction (10 ul volume)
My HK gene usually less than 10
I measured thre cDNA using nanaodrop
i found that >2000ng/ul
Any recommendation ?
You can diluated your cDNA. İts very intensity. you can try 1000 500 250 125 in one run and prefer best.
Hi Fatma
2000ng/ul is too high to have proper reactions in a little volume. molecules need to circulate and hybridize without to be space blocked. step dilutions are therefore necessary to approach the 100ng concentration. times ago I used 10ng by reactions, but depends on the level of your genes, try 100ng for all your samples.
fred
Dear Fatma,
Measuring cDNA concentration using nanodrop is not recommended, as primers (Oligo dT and Random primers) and even nucleotides used in cDNA synthesis have absorption at 260 nm.
Would you please let us know what your reference gene and GOI are? And also on what type of cell or tissue you are working?
Best,
Maryam
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