You can directly go for usual TriZol method. Since it is tissue plasma, which always a rich in miRNA contents as well as in plasma miRNA stays highly stable.

Total RNA-TriZol method from breast cancer cells, We are doing for a year, it works wonderfully!!!

Isolate total RNA....

(if your plan is gene expression then go for miRNA based cDNA synthesis using Qiagen miRcurry RT kit II, this kit convert all your miRNA from total RNA to miRNA-cDNA) and for gene expression.

I think following link may be helpful for you

I've had fairly good success with the miRNeasy qiagen kits.

Don't be put off by 'low yields': low yields are exactly what you'd expect because there simply isn't much RNA in plasma. I typically do not bother to spec mine at all, and simply us a consistent volume for downstream cDNA synthesis.

Remember: miRs are very small, so even a tiny amount of RNA (in ng) corresponds to a huge number of microRNAs.

Thank you all.

I have a cost limit to my thesis. I've bought a kit (GeneAll) for RNA extraction and I can not use another kit unless I use the appropriate manual method.

Please help me to improve RNA concentration.

Best Regards

Again, I am not sure you need to.

You haven't listed what your concentrations ARE, but remember that miRs are small. An mRNA for even a modest 30kDa protein is going to be ~1000 bases in length, but for the same mass you could get 50 microRNA molecules.

Even 1ng of RNA is ~100 billion microRNAs.

The other danger with trying to concentrate your RNA is that miRs (being small) are poorly precipitated, so clean-up/concentration steps tend to incur heavier losses from your miR population than might otherwise be expected.

If you are going for total RNA-TriZol method,

This few steps might help in improving total RNA extraction.

1. Try to process the the sample immediately

2. Use RNA Later or stabilizing solution to avoid degradation

3. Can keep overnight at -20C for precipitation

4. Resuspend in Pure TE buffer

If possible according to your conditions, you may increase cell number while going for RNA.

cheers!!!

Hi

U can use cell to ct kit.or mirnasy kit.

Hi,

Unfortunately, I could not find a solution for my problem, yet.

I upload some results, please guide me o find the source of the problem.

Thanks

Dear Maryam,

#1. Are you using Total RNA or Kit based isolated miRNA?

#2. What is concentration of RNA or isolated miRNA for cDNA synthesis and qPCR?

#3. What is cDNA process? Is it usual CDNA synthesis or miRNA specific cDNA synthesis

#4. what Primer you are using?

if you share these details, it will help to understand the issue?

Thanks

hope this link is useful

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3655275/

regards

900ng.ul is not a low yield: that's actually a really good yield for plasma.

Even if only a fraction of that is miRs, you should be seeing something, which suggests your cDNA synthesis is the stumbling block. What is your RNA 260/230 ratio? This should be high (ideally 2.0+ but at least 1.7 or more): low 260/230 ratios are indicative of salt contamination, and this can seriously hurt your cDNA synthesis efficiency.

Also, how much RNA are you using to make your cDNA, and how much of this cDNA are you then using in each well?

Thanks all. Because of high value of ct I think this results from nanodrop is not valid. Am I right?

Dear Maryam,

It could one of the possible reasons, when I started miRNA work, got similar high ct (~36) values.

I worked on these parts,

#1. Preferred to use in between 500-1000ng conc. Total RNA.

#2. I used qiagen miRcurry RT kit II to convert all my present miRNA in total RNA to miRNA-cDNA

#3. Since this above kit prepared cDNA worked suitable for SYBR so, SYBR green qPCR

#4 I used miRCURY LNA miRNA primer.

Now my getting in between ct 26-29 (depend on study), even respective downstream experiment and exiting reports in databases supporting the results.

here for me cDNA synthesis was crucial part, even cDNA synthesis kit contains spike-in component which enhances efficiency.

All the best!!!

With trizol you can extract total Rna which can be used for real time PCR. The concentration is 100 to 200 and Od about 1.4.