.................avoid using very small volumes for dilutions, errors increase greatly!Is the dilution that is wrong. You are using a 1 ng/ul instead of 10. 1 ul of 498ng/microliter= 489 ng. 489ng/489 ul total volume= 1 ng/ul. My advice here is to check your counts and avoid using very small volumes for dilutions, errors increase greatly!
I used 10ng total RNA for miRNA RT reaction (according to Taqman protocol, the amount of RNA is 10ng in each 15ul RT reaction).Because the concentrations of my total RNA isolated from Huh7-mock and Huh7-HCV replicon are 498ng/microliter and 520ng/microliter, I diluted total RNA to 10 ng/microliter by adding 1 microliter total RNA to 497 and 519 microliter RNase-free water,respectively.What's wrong with the dilution?
What is the difference between these two cell lines--one expresses the miRNA?
It should not matter much whether you dilute your RNA or not as long as you use the same amount of RNA for your RT reaction. However, the amount of RNA should be in a reasonable range. It seems that the amount of RNA you used is very low and the RNA was too diluted. Can you tell use the exact amount of RNA you used in the RT reaction?
thanks Fizban, but how should I dilute my total RNA?
What is your advice?
Thank you very much.