I was running RT-PCR for miRNA expression analysis from mice tissue last week but i get nothing in there.. I write detail of experiments. Guide me if i have made a mistake.
1. Total RNA Isolation trizole. very high concentration with almost 260/280 of around 1.98 each sample. so i diluted with dd water to make possible for pippting
2. 1st Strand Synthesis through Clontech Kit(Provided by Takara company here in China. Mir-x First Strand Synthesis KIT)
3. Then used Syber Premix Ex taq II for qPCR
What can be possible reason. waiting for Suggestion and your corrections.. Thanks.
Yes @ Hua Xiao You are right. the RNA looks viscous that is right; i didn't diluted du e to viscosity as some RNA concentration was low and not viscous, i did that to be able to pipet out a considerable amount. as if concentraion is too high then you have to take a very small amoutn of RNA to add for making cDNA.. so i diluted 10 times 1st then proceeded to cDNA, i have almost 15 RNA samples and its looks imposible that all RNA extracted have some Problem in extraction. and today i am going to Run some other Genes from the same RNA using normal cDNA procedure and RT-qPCR; that will show if there was a problem in RNA or not. More specifically it is not the 1st time i am dealing with RNA and making cDNA's but dealing with miRNA RT-qPCR is 1st time.
How much RNA are you using? What are the measured concentrations?
What are your 260/230 ratios?
(260/280 is a measure of protein contamination: has nothing to do with RNA concentration nor does it usually make much difference)
How are you designing your primers? Or are they bought from the same supplier that makes your miR cDNA synthesis kit?
Your positive controls both appear to have worked, which would tend to imply you have cDNA, but you haven't listed whether you DNAse treated your RNA before cDNA synthesis: U6 has many copies on the genome and these may well be amplified if you have genomic DNA contamination. Possibly sno65 would as well (might be worth doing a -RT control to see).
It would also help if you had a positive control for an actual miR, one that you know is expressed to high level in your tissue.
260/280 values are all around 1.9 while 260/230 are different but all above 1 and below 2. i didn't treated with DNAase. Primers were from another Recntly Published Paper; designed by sepaarate ompany from that of cDNA Kit. as the One Providing Kit don't have primer facility. Local Comapny Takra provided the KIT which is Clontec Supplier. i doubt the RNA as well now. i am going to Run RNA on Gel and if good then next time i will treat with DNAase too. good option too. following is the imags of RNA conetration as well as 60/280 and 260/230 values of RNA Samples.. Thanks for guidlines
Hi
Simple way is change ir primer stock.
If still ur not getting then check that any wrong programme put on real time machine I mean by mistake.u can check the CDna just fo regular pc5 and run it on gel.hope u have not changed the reagent if yes then check its compatibility with the assay ur using.i feel u can also check rox passive reference some time people keep syber green to syber green.instead u gave to keep syber green to rox.hope that sorts problem
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA
- microRNA