Do you have total RNA? I have obtained such a high concentration using traditional methods for RNA extraction (with TRIZOL reagent), when using commercial kits the concentration of total RNA is generally lower (10-20 ng/uL). I recommend you to check RNA integrity and purity with gel electrophoresis and/or spectrophotometer before proceeding with RT-PCR.
I think 1 ug of total RNA is OK for RT, actually if your mRNA transcript of interest is expected to have a low expression is better to use 1 ug for RT and cDNA generation.
But the optimal concentration for the qPCR assays is 20-50 ng of cDNA, so you will have to make a dilution of your sample.
Hope this helps you.
Best,
Check the volume required for the Reverse transcriptions step (max is normally 10ul) and dilute accordingly.
what source did you get your RNA from ?
Maybe be you have DNA contamination which is causing the high concentration ?
Hi Anupama,
1ug/ul of total RNA is not uncommon depending on the number of cells used for the extraction and the cell type as some cells are more abundant in RNA than others.
I would suggest to dilute your RNA based your concentration to achieve approximately 200-300 ng/ul and do the quantification again. This will be more accurate for the calculation and the pipetting for the reverse transcription.
Best,
Damien
Dear Castello,
Ya I isolated using Trizol but unusually I dint get sharp 2 bands rather a single blunt highly intense band appeared. Yes , I will go for dilution accordingly.
Dear Asif,
RNA is from typical bacterial slimy biofilm. So it was not easy to get transparent layer after trizol addition and centrifugation.
Dear Lawrence,
Starting material was high enough which might be the reason for such high quantity. I used 30 ul of water for dissolving.
Thanks all of you for your valuable reply :)
With Trizol total RNA isolation (in my case using 0.1 g lung tissue per 1 mL Trizol), I always get values of between 900 and 1500 ng/uL (total RNA/uL) in the end. And that is using 170 uL of nuclease-free water at the end for redissolving the total RNA pellet.
From there, after DNAse treatment, I have to dilute the samples at least 1:100 more post DNase treatment before the mRNA targets in them suitably amplify within the log linear range for all of my qPCR targets of interest (in a one-step RT-qPCR application).
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