I am measuring levels on mirco RNAs in a murine myoblast cell line. So, I collected the cells and processed them for small RNA extraction using MirVana Kit. Upon estimating the concentration and purity (260/280) using the nanodrop machine, I received a negative concentration value for most samples with poor purity.
I was supposed to go ahead with making cDNA and running it through qPCR to analyse the levels. I have two questions -
1. What could have gone wrong? This is the first time I am receiving such values.
2. Is it worth going ahead with cDNA and qPCR?
Dear Saurab
Same kind of problem I was also faced.
Isolate small RNA is little difficult due to very short not length.
Instead of Small RNA , I suggest you try to Total RNA isolation from your cell line using miRVana kit. You will get 260/280 ratio 2.0.
Because for cDNA conversion, you may require X concentration of Total RNA.( eg. 10-25 ng).
Pp
Prabhu, the kit comes with a total RNA extraction and further gives the steps for small RNA extraction. On my previous attempts, I used to perform total RNA extraction followed by small RNA and the results were fine. I used to get a small RNA concentration of ~ 30 ng/uL. But, this time it went awry. Just so you know, I have kept everything else - the kit, the reagents, my technique - steady.
Dear Saurabh
I am using Exiqon miRNA universal cDNA kit, they recommended 10ng of total RNA use to convert cDNA.
May I know your cDNA kit recommend whether small RNA or total RNA concentration to be used to convert cDNA.
Pp
Prabhu, thanks for following up. I am using TaqMan MicroRNA Assays kit by applied biosystems. The recommendations state that the kit is sensitive enough and requires between 1 and 10 ng of total RNA or equivalent. What do you make of it?
Dear Saurabh
My suggestion is use Total RNA. I am working in the microRNA field for last 5 years.
Prabhu, so that would mean stopping at the total RNA extraction step and proceeding with the small RNA purification protocol. Fair enough. I will give it a go.
Dear Saurabh
My suggestion is
1. Proceed Isolate Total RNA procedure.
2. For cDNA conversion use Total RNA (concentration 1 - 10 ng) as per you kit.
3. Please check your TaqMan® MicroRNA Reverse Transcription Kit ( whether Total RNA or small RNA).
If it possible can you send me TaqMan® MicroRNA Reverse Transcription Kit Manual.
If any further clarification please
Best wishes
pprabu
Saurabh,
As you are using a spectrophotometer to measure your sample, you'd better use 20 ng of RNA to prepare your cDNA, because the spectrophotometer for such small quantities is not the best method of quantitation.
hi saurabh,
I don`t know any answer about your question.but I have a question .Taqman MicroRNA assay is used in your research .I want to use applied biosystems products .Unfortunately, I have just a few information about them. Could you give me some information about assay ID (382)?Is this assay ID consist of primers and taqman prob(for real time) except of stem loop primer for RT?Thanks.
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